Compositions and Methods for Gene Silencing

ABSTRACT

Compositions and methods for modulating the expression of a protein of interest are provided.

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 13/687,644, filed on Nov. 28, 2012, which is acontinuation application of U.S. patent application Ser. No. 13/082,865,filed on Apr. 8, 2011, now U.S. Pat. No. 8,343,941, which is acontinuation-in-part of U.S. patent application Ser. No. 12/570,389,filed on Sep. 30, 2009, which claims priority under 35 U.S.C. §119(e) toU.S. Provisional Patent Application No. 61/144,087, filed on Jan. 12,2009, and is a continuation-in-part of PCT/US2008/058907, filed on Mar.31, 2008, which claims priority under 35 U.S.C. §119(e) to U.S.Provisional Patent Application No. 60/921,032, filed on Mar. 30, 2007.This application also claims priority under 35 U.S.C. §119(e) to U.S.Provisional Patent Application No. 61/898,145, filed on Oct. 31, 2013.The foregoing applications are incorporated by reference herein.

This invention was made with government support under Grant No. CA153842awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

This invention relates generally to the field of gene silencing.Specifically, the invention provides compositions and methods forregulating the expression of a gene of interest.

BACKGROUND OF THE INVENTION

It has long been appreciated that gene expression can be regulated atthe post-transcriptional level, defined to be the steps betweentranscription initiation and release of the nascent polypeptide from theribosome. Antisense based approaches encompass a broad variety oftechniques, but have in common an oligonucleotide that is designed tobase pair with its complementary target mRNA, or more broadly to anyRNA, leading to either degradation of the RNA or impaired function(e.g., impaired translation). Classical antisense approaches weredesigned to interfere with translation of the target mRNA or induce itsdegradation via Rnase H. Ribozyme-containing antisense molecules alsocan induce RNA degradation and have the advantage that they can beturned over (i.e., re-used) to cleave more RNA targets. RNAi-basedapproaches have proven more successful and involve using siRNA to targetthe mRNA to be degraded (see, e.g., Novina et al. (2004) Nature430:161-4). However, some mRNAs are only modestly downregulated (2-fold)by RNAi and others are refractory.

The 3′ end processing (also called polyadenylation, poly(A) tailaddition, or cleavage and polyadenylation) of nearly all eukaryoticpre-mRNA comprises two steps: (1) cleavage of the pre-mRNA followed by(2) the synthesis of a poly(A) tail at the 3′ end of the upstreamcleavage product. 3′ end formation is essential to mRNA maturation and,in this sense, is as important as transcription initiation for producinga functional mRNA. 3′ end formation also functions to enhancetranscription termination, transport of the mRNA from the nucleus, andmRNA translation and stability (Eckner et al. (1991) EMBO J.,10:3513-3522; Sachs et al. (1993) J. Biol. Chem., 268:22955-8). Defectsin mRNA 3′ end formation can profoundly influence cell growth,development and function (see, e.g., Zhao et al. (1999) Microbiol. Mol.Biol. Rev., 63:405-445; Proudfoot et al. (2002). Cell 108:501-12).

Cleavage and polyadenylation requires two elements. The highly conservedAAUAAA sequence (also called the poly(A) signal or the hexanucleotidesequence) is found 10 to 30 nucleotides upstream of the cleavage site.This hexanucleotide is essential for both cleavage and polyadenylationand any point mutations (with the exception of AUUAAA) result in a largedecrease in its activity (Proudfoot et al. (1976) Nature 263:211-4;Sheets et al. (1990) Nucl. Acids Res., 18:5799-805). However, recentbioinformatic studies have suggested that single-base variants ormore-rarely double-base variants of AWUAAA (W=A or U) are allowed(Beaudoing et al. (2000) Gen. Res. 10:1001-1010; Tian et al. (2005) Nuc.Acids Res., 33:201-12).

The second element is a less-conserved U- or GU-rich regionapproximately 30 nucleotides downstream of the cleavage site and thus iscalled the downstream sequence element (DSE). Point mutations or smalldeletions do not greatly influence DSE's function. Nevertheless, theproximity of the DSE to the poly(A) site can affect the choice of thecleavage site and the efficiency of cleavage (Zhao et al. (1999).Microbiol. Mol. Biol. Rev., 63:405-445). The cleavage site itself(usually referred to as the pA site or poly(A) site) is selected mainlyby the distance between the AAUAAA signal and the DSE (Chen et al.(1995). Nuc. Acids Res., 23:2614-2620). For most genes, cleavage happensafter a CA dinucleotide.

In addition to the above signals, auxiliary sequences have also beenfound to have a positive or negative modulatory activity on 3′ endprocessing.

The cleavage/polyadenylation machinery is composed of multiple proteinfactors with some having multiple subunits. The endonucleolytic cleavagestep involves Cleavage/Polyadenylation Specificity Factor (CPSF) bindingto A(A/U)UAAA and Cleavage stimulatory Factor (CstF) binding the DSE.Other required factors include Cleavage Factors 1 and 2 (CF I_(m) and CFII_(m)), RNA polymerase II (Pol II), Symplekin, and poly(A) polymerase(PAP), although the absolute requirement for PAP is still unclear. Oncecleavage has occurred the downstream pre-mRNA fragment is rapidlydegraded whereas the upstream fragment undergoes poly(A) tail additionthat requires CPSF, PAP, and poly(A)-binding protein II (PAB II).

In principle, having the ability to switch on or off a gene's poly(A)site or sites is a way to directly control expression of that gene.There are natural examples where a gene's expression can be controlledby dialing up or down the poly(A) site. Perhaps the best understoodexample involves excess U1A protein negatively autoregulating its ownsynthesis by inhibiting polyadenylation of its own pre-mRNA. Without apoly(A) tail, the mRNA fails to leave the nucleus and is degradedleading to lower levels of U1A mRNA and U1A protein. The mechanisminvolves 2 molecules of U1A protein binding to a site just upstream ofits own pre-mRNA's poly(A) site with the resulting (U1A)₂-pre-mRNAcomplex inhibiting 3′-end processing of the U1A pre-mRNA by inhibitingthe polyadenylation activity of PAP (Boelens et al. (1993) Cell72:881-892; Gunderson et al. (1994) Cell 76:531-541; Gunderson et al.(1997) Genes Dev., 11:761-773). An illustrative example, albeitartificial, of “dialing” is found in Guan et al. (Mol. Cell. Biol.(2003) 23:3163-3172). Guan et al. demonstrate that endogenous U1Aprotein levels are dialed up or down by dialing up or down the activityof its poly(A) site through a stably-expressed epitope-tagged U1Aprotein that is under the control of a Tet-regulated promoter. Theepitope-tagged U1A protein is not subject to autoregulation because itsexpression cassette lacks the autoregulatory 3′UTR element.

Another natural example of dialing a poly(A) site involves U1 snRNPbinding to a “U1 site” just upstream of the poly(A) site of the bovinepapillomavirus type 1 (BPV1) late gene pre-mRNA (Furth et al. (1994)Mol. Cell. Biol., 14: 5278-5289). The term “U1 site”, which stands forU1 snRNP binding site, is used so as to distinguish it from U1 snRNP'sbetter known function in 5′ splice site (5′ss) binding during pre-mRNAsplicing. U1 snRNP consists of 10 proteins in complex with a 164nucleotide U1 snRNA that base pairs to the BPV1 U1 site via nucleotides2-11 of U1 snRNA (see, e.g., Will et al. (1997) Curr. Opin. Cell Biol.,9:320-8), notably the same nucleotides 2-11 also basepair to the 5′sssequence as part of the splicing mechanism. Subsequent to its discoveryin BPV1, mechanistic studies demonstrated the U1-70K component of the U1snRNP directly binds to and inhibits the polyadenylation activity ofpoly(A) polymerase (Gunderson, et al. (1998) Mol. Cell 1:255-264), theenzyme that adds the poly(A) tail. Additional studies in vivo thateliminated the U1-70K binding site confirmed U1-70K as the effectorsubunit that inhibits expression (Beckley et al. (2001) Mol. Cell Biol.,21:2815-25; Sajic et al. (2007) Nuc. Acids Res., 35:247-55). More recentstudies have demonstrated U1 snRNP's polyA site inhibitory activity ispart of a broader “surveillance” function that it has in quality controlof RNA processing, in particular to suppress internal polyA sites thatwould otherwise lead to truncated mRNAs (Kaida et al., Nature (2010)468:664-8; Berg et al., Cell (2012) 150:53-64).

The U1in gene silencing technologies use 5′-end-mutated U1 snRNA (see,e.g., U.S. Patent Application Publication Nos. 2003/0082149 and2005/0043261). U1in stands for U1 snRNP inhibition of expression andrefers to two recently developed gene silencing technologies thatinvolve expression of a 5′-end-mutated U1 snRNA where nucleotides 2-11of U1 snRNA are complementary to a 10 nucleotide sequence in the targetgene's 3′ terminal exon. The 5′-end-mutated U1 snRNA is expressed from aU1 snRNA expression cassette containing promoter elements and a 3′ endformation signal from the U1 snRNA gene. The 5′-end-mutated U1 snRNAtranscript assembles with the canonical U1 snRNP proteins into a5′-end-mutated U1 snRNP that then binds to and inhibits polyadenylationof the targeted pre-mRNA. The 3 key features to make U1in silencing workare: (1) the U1 site on the target pre-mRNA and the 5′-end-mutated U1snRNA must be perfectly complementary across all 10 basepairs, as asingle base mismatch is sufficient to lose silencing (Liu et al. (2002)Nuc. Acids Res., 30:2329-39), (2) the U1 site must be in the 3′ terminalexon of the target pre-mRNA (Beckley et al. (2001) Mol. Cell Biol.,21:2815-25; Fortes et al. (2003) Proc. Natl. Acad. Sci., 100:8264-8269),and (3) the U1-70K binding site on the U1 snRNA must be intact. AlthoughU1in has been successfully used in several instances, its development asa widely-used technology has been limited for a variety of reasons.

In view of the foregoing, it is clear that there is still a need formethods of regulating gene expression.

SUMMARY OF THE INVENTION

In accordance with the instant invention, nucleic acid molecules forinhibiting the expression of a gene of interest are provided. In aparticular embodiment, the nucleic acid molecules comprise an annealingdomain operably linked to at least one effector domain, wherein theannealing domain hybridizes to the pre-mRNA of the gene of interest andwherein the effector domain hybridizes to the U1 snRNA of U1 snRNP.

In accordance with another aspect of the instant invention, the nucleicacid molecules may be conjugated to (e.g., directly or via a linker) atargeting moiety. The targeting moiety may be conjugated to the 5′ endand/or the 3′ end (e.g., the nucleic acid may comprise two targetingmoieties, either the same or different). In a particular embodiment, thenucleic acid molecules are conjugated to an aptamer.

In accordance with another aspect of the invention, methods are providedfor inhibiting the expression of a gene of interest comprisingdelivering to a cell at least one of the nucleic acid molecules of theinstant invention.

In accordance with another aspect of the invention, compositions areprovided which comprise at least one of the nucleic acid molecules ofthe invention and at least one pharmaceutically acceptable carrier.

In still another aspect, vectors encoding the nucleic acid molecules ofthe instant invention are also provided.

In accordance with another aspect of the instant invention, methods oftreating, inhibiting, and/or preventing a cancer in a subject areprovided.

BRIEF DESCRIPTIONS OF THE DRAWING

FIG. 1A is a schematic of a U1 adaptor oligonucleotide depicting its 2domains: an annealing domain to base pair to the target gene's pre-mRNAin the 3′ terminal exon and an effector domain that inhibits maturationof the pre-mRNA via binding of endogenous U1 snRNP. The providedsequence of the effector domain is SEQ ID NO: 1. FIG. 1B is a schematicof the U1 adaptor annealing to target pre-mRNA. FIG. 1C is a schematicof the U1 adaptor binding U1 snRNP, which leads to poly(A) siteinhibition. Ψ=pseudouridines of the U1 snRNA in the U1 snRNP. Theprovided sequence of the U1 snRNA in the U1 snRNP is SEQ ID NO: 2.

FIG. 2A provides schematics of (1) p717B, comprising a standard Renillareporter with its 3′UTR and poly(A) signal sequences replaced with thosefrom the human MARK1 gene that has a naturally occurring U1 site (SEQ IDNO: 1), and (2) p717ΔB, which matches p717B except for a 4 nucleotidemutation (lowercase letters in SEQ ID NO: 4) in the U1 site. Relativeexpression levels of the plasmids upon transient expression in HeLacells along with a Firefly luciferase control are shown, indicating thewild type U1 site represses expression by 30-fold. FIG. 2B provides aschematic of a U1 adaptor inhibiting Renilla luciferase expression. Thep717ΔB Renilla reporter with a MARK1 3′UTR having a mutated U1 site (SEQID NO: 5) was co-transfected with LNA6 (SEQ ID NO: 6), a U1 adaptordesigned to inhibit the poly(A) site via binding of endogenous U1 snRNP(SEQ ID NO: 2). The bold font indicates LNA bases to increase annealing.LNA7 (SEQ ID NO: 7) is a control that matches LNA6 except for mutationof the effector domain. The LNA6 and LNA7 binding site is indicated bythe shaded box.

FIG. 3A is a graph of the inhibitory activity of LNA6 and LNA7 on theRenilla reporter. Grey bars are LNA6, white bars are LNA7, and the blackbar is M13. FIG. 3B provides a graph of the inhibitory activity of LNA6as a function of concentration. Values are normalized to the M13 controloligo. IC₅₀=Inhibitory Concentration needed to achieve 50% inhibition.The bottom curve is the inhibition of p717ΔB and the top curve is theinhibition of the SV40 reporter having the 15 nucleotide isolated LNA6binding site (gray box in FIG. 3C). FIG. 3C provides schematics ofp717ΔB and pRL-LNA6.

FIG. 4A provides schematics of pRL-LNA6 and pRL-(LNA6)₂. FIG. 4Bprovides a graph demonstrating the inhibitory activity of LNA6 U1adaptor on plasmids containing one or two LNA6 binding sites. Values arenormalized to the LNA7 control oligo. The top curve is the inhibition ofthe pRL-LNA6 plasmid and the bottom curve is the inhibition of thepRL-(LNA6)₂ plasmid.

FIG. 5A is a schematic of a U1 adaptor (LNA13; SEQ ID NO: 13) designedto target the C-raf-1 pre-mRNA by targeting a 3′UTR sequence. The boldfont indicates LNA bases to increase annealing. The U1 snRNP sequence isSEQ ID NO: 2 and the flanking and LNA13 binding site is SEQ ID NO: 12.FIG. 5B is a graph depicting the inhibitory activity of varyingconcentrations of LNA13 on C-raf-1 mRNA as measured by Q-PCR andnormalized to GAPDH.

FIG. 6A provides a schematic of the Renilla reporter pRL-wtC-Raf-1 (alsocalled p722L) with a C-raf-1 3′UTR and sequences past the poly(A) site,and a graph depicting the IC₅₀ values for LNA13 inhibition ofpRL-wtC-Raf-1 expression. FIG. 6B provides a schematic of plasmidpRL-LNA13 which has a single binding site for LNA13, and a graphdepicting the inhibition of expression by LNA13 as a function ofconcentration.

FIG. 7A provides the sequences of LNA6 (SEQ ID NO: 6), LNA17 (SEQ ID NO:14), Ome-1 (SEQ ID NO: 15), and Ome-5 (SEQ ID NO: 16). FIG. 7B providesa graph of the inhibitory activity of 60 nM of adaptors co-transfectedwith the pRL-wtC-raf-1 plasmid or the pRL-SV40 control plasmid into HeLacells.

FIG. 8A provides the sequences of LNA17 (SEQ ID NO: 14), LNA21 (SEQ IDNO: 17), LNA22 (SEQ ID NO: 18), and LNA23 (SEQ ID NO: 19). FIG. 8Bprovides a graph of the inhibitory activity of adaptor variants of LNA17having phosphorothioate (PS) bonds and different attachment sites forthe U1 domain by co-transfection with pRL-LNA6 (p782J) in HeLa cells.

FIG. 9A provides the sequences of LNA17 (SEQ ID NO: 14), LNA24/15 (SEQID NO: 20), and LNA24/12 (SEQ ID NO: 21). FIG. 9B provides a graphdepicting the inhibitory activity of LNA17, LNA24/15, and LNA24/12.

FIG. 10 provides a schematic of pRL-wtC-raf-1 and the sequences of LN13(SEQ ID NO: 13) and LNA25-mtH/U1 (SEQ ID NO: 22). The inhibitoryactivity of 30 nM of adaptors co-transfected with the pRL-wtC-raf-1plasmid into HeLa cells is also provided.

FIG. 11A provides a schematic of p782J and the sequences of LNA6 (SEQ IDNO: 6), LNA17-13 (SEQ ID NO: 23), LNA17-12 (SEQ ID NO: 24), LNA17-11(SEQ ID NO: 25), LNA17-10 (SEQ ID NO: 14), LNA17-9 (SEQ ID NO: 26),LNA17-8 (SEQ ID NO: 27), and LNA17-7 (SEQ ID NO: 28). FIG. 11B providesa graph of the inhibitory activity of 30 nM of adaptors co-transfectedwith the pRL-LNA6 plasmid into HeLa cells.

FIG. 12 provides a schematic of pRL-LNA6 and a graph depicting theinhibitory activity of LNA17-11 adaptor when transfected into differentcells lines. DU145 and PC3 are human cell lines originally derived frommore aggressive prostate cancers, whereas LnCap was derived from a lessaggressive prostate cancer. SH-SY5Y is a human brain cell line.

FIG. 13A provides a graph depicting the inhibition activity with thecombination of co-transfected siRNA and U1 adaptors. FIG. 13B provides aschematic of pRL-GADPH and the sequence of LNA12 (SEQ ID NO: 29). FIG.13C provides a graph depicting the inhibition activity of thecombination of co-transfected siRNA and U1 adaptors.

FIG. 14A provides a schematic of p722L and the sequences of LNA-mtH/U1(SEQ ID NO: 22), LNA25-H/mtU1 (SEQ ID NO: 30), and LNA25-H/U1 (SEQ IDNO: 31). FIG. 14B provides a graph depicting the inhibitory activity ofthe combination in one oligonucleotide of U1 Adaptor activity and RnaseH activity (i.e., traditional antisense design).

FIG. 15A provides images of Western blots of 40 μg of total proteinextracts from HeLa cells transfected in 6 well plates with 30 nMoligonucleotide. The proteins were separated on 12% SDS-PAGE and probedwith mouse cRAF, PARP, and GAPDH antibodies. FIG. 15B provides a graphdepicting the Q-PCR with C-raf-1-specific primers with the datanormalized to GAPDH mRNA.

FIG. 16A provides Western blots of transfected HeLa cells lysed in SDSbuffer and probed with mouse cRAF and GAPDH antibodies. FIG. 16Bprovides a graph depicting Q-PCR with C-raf-1-specific primers with thedata normalized to GAPDH mRNA.

FIGS. 17A-17D demonstrate U1 Adaptor inhibition of Renilla is at thelevel of reduced mRNA. FIGS. 17A and 17B are images of RPA analysis of 3μg total RNA from untransfected HeLa cells or co-transfected withpRL-UA6 and 30 nM of each oligonucleotide, either the M13 control, theUA6 Adaptor, the UA7a control Adaptor or the UA17-13 Adaptor, asindicated. After 24 hours, the cells were harvested and split into twoportions, one to measure Luciferase and the other to make total RNA usedfor RPAs with either a Renilla-specific probe (FIG. 17A lanes 1-10) or aGAPDH-specific probe (FIG. 17B lanes 1-9). Note that Lanes 1-8 and 10 ofFIG. 17A are the same total RNA samples as lanes 1-8, and 10 in FIG.17B. Lane 9 is blank so as to separate the stronger signal in lane 10from lane 8. The lanes marked “Msp Marker” are a ³²P-end-labeled Mspdigest of pBR322 with the sizes of the bands indicated. The lane marked“cytoplasmic M13” is total RNA from the cytoplasmic fraction of M13transfected cells prepared as described (Goraczniak et al. (2008) J.Biol. Chem., 283:2286-96). FIG. 17C is an image of an RPA analysis as inFIG. 17A, but with varying amounts of total RNA as indicated. FIG. 17 Gis a graph of the quantitation of RPA protected bands and a comparisonwith the corresponding Renilla Luciferase activities. Both the RPA andLuciferase values were normalized to M13 that was set to 100%.

FIG. 18 is an image of the detection by EMSA of the UA6 Adaptortethering U1 snRNP to the target RNA. A ³²P-uniformly labeled RNA (˜300nt), called UA6 RNA, was made by T7 RNA Polymerase in vitro run offtranscription from a PCR template amplified from pRL-UA6 containing theUA6 binding site. ³²P-UA6 RNA (1 pmol) was mixed either with highlypurified HeLa cell U1 snRNP, a U1 Adaptor, or both and the resultingcomplexes resolved by 6% native PAGE in 1 xTBE containing 5% glycerol.The purification of U1 snRNP and its use in EMSA was previouslydescribed (Abad et al. (2008) Nucleic Acids Res., 36:2338-52; Gundersonet al. (1998) Molecular Cell, 1:255-264).

FIG. 19 provides a graph which shows the affect of increasing the lengthof the U1 Domain. The UA17 Adaptor series has the same Target Domain asthe UA6 Adaptor and has a U1 Domain made of 100% 2′OMe RNA. Length ofthe U1 Domain varies from 7 nts (UA17-7 Adaptor) to 19 nts (UA17-19Adaptor). LNA nucleotides are bold uppercase, DNA nucleotides areunderlined uppercase, and 2′OMe RNA nucleotides are lowercase. 15 nM ofeach U1 Adaptor was co-transfected with pRL-UA6 into HeLa cells andinhibitory activities calculated. The UA7b and UA7c Adaptors arenegative controls bearing a single (7b) or double (7c) mutation in theU1 Domain. The sequences provided are SEQ ID NO: 6, SEQ ID NO: 34, SEQID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 23, SEQ ID NO: 24,SEQ ID NO: 25, SEQ ID NO: 14, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO:28, SEQ ID NO: 38, and SEQ ID NO: 39, from top to bottom.

FIG. 20 demonstrates the activity of various UA17 Adaptors correlateswith their affinity to U1 snRNP. Shown is an EMSA-based competitionassay for U1 snRNP binding between various unlabeled UA17 Adaptors andthe ³²P-U1D-RNA having an 11 nt U1 Domain (5′CAGGUAAGUAU3′; SEQ ID NO:32). 0.75 pmoles of purified HeLa cell U1 snRNP was mixed with 0, 1 or 3pmoles of various unlabeled UA17 Adaptors (the competitor) and incubatedfor 20 minutes at 30° C. Next 0.5 pmoles of ³²P-labeled U1D-RNA wasadded and incubated for 10 min at 30° C. and then the complexes resolvedby native PAGE. Lane 1 contains no U1 snRNP and no competitor whereaslane 2 contains no unlabeled UA17 competitor Adaptor. The U1snRNP:³²P-U1D-RNA complex in lane 2 was set to 100% as the reference.The numbers in the center of the autoradiogram are values relative tothe lane 2 reference. The panel on the left is a lighter exposure oflanes 1-4 so as to visualize that the UA17-7 competitor Adaptor had nodetectable effect on the amount of complex formation. The competitionassay was repeated 3× with similar results.

FIG. 21 provides a graph demonstrating the affect of placing the U1Domain at the 5′ end of the Adaptor. The UA22 Adaptor series matchesUA17 except the relative position of the U1 and Target Domains arereversed so that the U1 Domain is positioned at the 5′ end.Transfections and analysis are as in FIG. 19. The graph summarizes theresults of testing the UA22 series side-by-side with the correspondingUA17 series. The sequences provided are SEQ ID NO: 40, SEQ ID NO: 41,and SEQ ID NO: 42, from top to bottom.

FIG. 22 provides a graph demonstrating the affect of substituting LNAnucleotides into the U1 Domain. The inhibitory activity of UA17-13 (SEQID NO: 23), the most active U1 Adaptor from FIG. 19, was compared with amatching Adaptor UA17-13b (SEQ ID NO: 43), which has five LNAnucleotides in the U1 Domain.

FIG. 23 demonstrates the specificity assessed by a mutation/compensatorymutation analysis. Three U1 Adaptors, UA17-m1 (SEQ ID NO: 46), UA17-m2(SEQ ID NO: 45), and UA17-m3 (SEQ ID NO: 44), are shown that matchUA17-13b (SEQ ID NO: 43) except they have 1, 2 or 3 nt mismatches (inlighter font) to the target sequence in the pRL-UA6 “wild type” reporter(SEQ ID NO: 47). These three U1 Adaptors also have a slightly alteredconfiguration of LNA-DNA nucleotides when compared to UA17-13b which wasnecessary to avoid high self annealing scores that could potentiallyreduce activity. The activity of each of these four U1 Adaptors wasdetermined by transfection into HeLa cells with either the pRL-UA6reporter or the pRL-UA6-m3 reporter (SEQ ID NO: 48), the latterrestoring perfect complementarity to the UA17-m3 Adaptor. Twoconcentrations of U1 Adaptors were used (2.5 nM in the upper panel and 5nM in the lower panel) and the results are from 3 independentexperiments.

FIGS. 24A and 24B demonstrate that U1 Adaptors have no effect onsplicing of a reporter gene. FIG. 24A depicts pcDNA3.1+, a standardmammalian expression vector, which was modified by inserting a ˜3000 bpsegment of the human Fibronectin (FN) gene (spanning exons III7b toIII8a that includes ˜2300 nt intron). The 3′UTR and polyA site sequencesare derived from the bovine growth hormone gene. pFN-1for has a 15 ntUA6 binding site inserted in the forward orientation about ˜300 nts intothe intron while pFN-1rev has the UA6 binding site inserted at the sameposition but in the reverse orientation. pFN-2for and pFN-2rev are likepFN-1for and pFN-1rev, respectively, but the binding site was inserted˜270 nt upstream of the 3′ss boundary. pFN-3for and pFN-3rev are likepFN-1for and pFN-1rev, respectively, but the binding site was insertedin the terminal exon. pFN-4for and pFN-4rev are like pFN-1for andpFN-1rev, respectively, but the binding site was inserted in the firstexon. For FIG. 24B, each of the pFN plasmids was transfected into HeLacells either with 5 nM UA17-13b Adaptor (the most active U1 Adaptortargeting the UA6 binding site) or 5 nM M13 control oligonucleotide andafter 24 hours the cells were harvested and analyzed by RT-PCR. The T7and BGH primers were specific to the reporter as no band was detected inthe untransfected control cells (lanes 11 and 22). Also shown is theRT-PCR amplification of the endogenous Arf1 gene so as to control forthe quality of the RNA sample and the RT-PCR.

FIGS. 25A-25C demonstrate the inhibition of the endogenous RAF1 gene.FIG. 25A shows the design of the UA25 Adaptor (SEQ ID NO: 22) thattargets the human RAF1 gene. UA25-mt (SEQ ID NO: 49) is a controlAdaptor that matches UA25 except for a 2 nt mutation in the U1 Domain.Symbols are as in FIG. 19. FIG. 25 is an image of a Western blot with ananti-RAF1 antibody demonstrating the UA25 Adaptor specifically silencedRAF1 protein in a dose dependent manner when transfected into HeLacells. The same blot was striped and reprobed with anti-GAPDH antibodyto control for equal loading. The same set of transfected cells wassplit into two with one part being analyzed by Western blotting and theother by qPCR. FIG. 25 C is a graph of a qPCR analysis demonstratingthat RAF1 silencing by the UA25 Adaptor occurs at the mRNA level. qPCRwas performed and levels of RAF1 mRNA were normalized to the internalstandard GAPDH mRNA. Results in FIGS. 25B and 25C are from 3 independenttransfections.

FIGS. 26A and 26B demonstrate the inhibition of RAF1 with threedifferent anti-RAF1 U1 Adaptors. FIG. 26A shows the design of threeanti-RAF1 U1 Adaptors. FIG. 26B shows a Western blot of total celllysates (25 μg/lane) from cells transfected with 30 nM of the anti-RAF1U1 Adaptors using M13 oligonucleotide as a control. The sequencesprovided are SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, from topto bottom.

FIGS. 27A and 27B demonstrate the inhibition of the endogenous PCSK9gene and enhanced inhibition with multiple Adaptors. FIG. 27A shows thesequences of two anti-PCSK9 U1 Adaptors. FIG. 27B is a graph showing theanti-PCSK9 U1 Adaptors were transfected alone or together into HeLacells. After 24 hours total RNA was harvested and analyzed by qPCR tomeasure silencing of PCSK9. Results are from 3 independenttransfections. The sequences provided are SEQ ID NO: 53 and SEQ ID NO:54, from top to bottom.

FIGS. 28A and 28B demonstrate that the separation of the U1 and TargetDomains inactivates U1 Adaptors. Based on the UA17-13b (SEQ ID NO: 43)design (targeting Renilla Luciferase), two “half” Adaptors weresynthesized: UA17-13b-TD (SEQ ID NO: 55) has only the Target Domain (TD)and UA17-13b-U1D (SEQ ID NO: 56) has only the U1 Domain (U1D) (FIG.28A). Co-transfection of 5 nM of each half Adaptor alone or togethergave no significant inhibition of the pRL-UA6 reporter as compared tothe M13 control (FIG. 28A). In contrast, transfection of the UA17-13bAdaptor gave an 88% level of inhibition of Renilla in agreement withwhat was obtained previously. Based on the UA31e (SEQ ID NO: 54) design(targeting endogenous PCSK9), two “half” Adaptors were synthesized whereUA31e-TD (SEQ ID NO: 57) has only the Target Domain (TD) and Ua31e-U1D(SEQ ID NO: 58) has only the U1 Domain (U1D) (FIG. 28B). Co-transfectionof 5 nM of each half Adaptor alone or together gave no significantreduction of endogenous PCSK9 mRNA as compared to the M13 control (FIG.28B). In contrast, transfection of the UA31e Adaptor gave an 80% levelof inhibition in agreement with results shown previously.

FIGS. 29A and 29B demonstrate the co-transfection of U1 Adaptors and asiRNA results in enhanced silencing. FIG. 29A is a graph demonstratingthe co-transfection of the UA17-13b Adaptor and an anti-Renilla siRNA(RL-siRNA) with the reporter construct pRL-UA6 into HeLa cells givesenhanced silencing as compared to transfection of either the U1 Adaptoror the siRNA alone. pRL-UA6rev is a control plasmid where the U6 bindingsite is in the reverse orientation and so should not be inhibited by theUA17-13b Adaptor if inhibition occurs at the mRNA level. FIG. 29B is agraph showing that the co-transfection of the anti-RAF1 UA25 Adaptorwith an anti-RAF1 Dicer-substrate siRNA (DsiRNA) gives enhancedsilencing of the endogenous RAF1 gene as compared to transfection ofeither the U1 Adaptor or the siRNA alone. Western blotting to detectRAF1 confirmed enhanced inhibition is also seen at the protein level.Results in FIGS. 29A and 29B are from 3 independent transfections.

FIGS. 30A and 30B demonstrate that the co-transfection of U1 Adaptorsand siRNAs gives enhanced silencing. FIG. 30A shows the design of ananti-GAPDH UA12 Adaptor (SEQ ID NO: 29) and a Renilla reporter calledpRLGAPDH having its 3′UTR and polyA signal sequences derived from thoseof the human GAPDH gene. Transfection analysis of UA12's inhibitoryactivity on pRL-GAPDH expression gave an IC₅₀ of 1.8 nM (FIG. 30A).Co-transfection of the UA12 Adaptor and an anti-Renilla siRNA (RL-siRNA)with pRLGAPDH into HeLa cells gives enhanced silencing as compared totransfection of the U1 Adaptor or the siRNA alone (FIG. 30B). Thecontrol siRNA (Ctr-siRNA) had no effect.

FIGS. 31A and 31B demonstrate that the co-transfection of U1 Adaptorsand a siRNA gives enhanced silencing of the endogenous PCSK9 gene. FIG.31A shows the design of two anti-PCSK9 U1 Adaptors. Co-transfection ofthe two anti-PCSK9 U1 Adaptors and an anti-PCSK9 siRNA into HeLa cellsfor 24 hours gives enhanced silencing as compared to transfection of theU1 Adaptors or the siRNA alone (FIG. 30B). The sequences provided areSEQ ID NO: 53 and SEQ ID NO: 54, from top to bottom.

FIGS. 32A and 32B depict global expression analysis comparing U1Adaptors to siRNAs. The total RNAs from the M13, DsiRNA and U1 Adaptortransfections were analyzed by microarray with the Affymetrix humanU133A chip and the R2:R1 and R3:R1 ratios calculated for all the genes(FIG. 32A). The fold-reduction in PCSK9 levels obtained by microarray iscompared to the values obtained by qRT-PCR. FIG. 32B provides acomparison plot of the genes that showed ≧2-fold change for the R2:R1ratio (anti-PCSK9 DsiRNA) and the R3:R1 ratio (anti-PCSK9 U1 Adaptors).The line represents the ≧2-fold affected genes from the U1 Adaptortransfection that are sorted from the largest increase to the largestdecrease. The other lines represent the corresponding genes but from theDsiRNA transfection. If both the U1 Adaptor and siRNA methods wereperfectly specific then the lines would perfectly overlap.

FIG. 33 demonstrates that U1 Adaptors have no apparent effect onalternative splicing pattern of certain genes. The panels show fourRT-PCRs that detect alternative splicing of 4 endogenous genes (Cdc25B,Cdc25C, Grb2 and Fibronectin). For all panels, lanes 1-3 are the sameRNAs shown that were transfected with 5 nM M13 (lane 1) or 5 nM each ofUA31d4 and UA31e (lane 2) or 5 nM anti-PCSK9 siRNA (lane 3). UniformRT-PCR bands for the Arf1 housekeeping gene were observed for thesesamples demonstrating the RNA samples and the RT-PCR were of similarquality.

FIG. 34 provides a list of the U1 Adaptor sequences used in this work.The U1 Adaptor names, sequences and the target mRNA are indicated. TheU1 Domain is in lighter font, the Target Domain in black. U1 Adaptorsfrom the same series were aligned according to their Target Domains. Theasterisk indicates a matching Adaptor has been tested that has a 100%phosphorothioate backbone. All Adaptors were manufactured by IDT(Coralville, Iowa) and purified by HPLC prior to use. The sequencesprovided are SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 38, SEQ ID NO: 39,SEQ ID NO: 15, SEQ ID NO: 28, SEQ ID NO: 27, SEQ ID NO: 26, SEQ ID NO:14, SEQ ID NO: 25, SEQ ID NO: 24, SEQ ID NO: 23, SEQ ID NO: 43, SEQ IDNO: 37, SEQ ID NO: 36, SEQ ID NO: 35, SEQ ID NO: 34, SEQ ID NO: 44, SEQID NO: 45, SEQ ID NO: 46, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 21,SEQ ID NO: 59, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO:60, SEQ ID NO: 22, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ IDNO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 58, SEQID NO: 55, SEQ ID NO: 56, SEQ ID NO: 29, and SEQ ID NO: 61, from top tobottom.

FIG. 35 provides a list of the siRNA sequences used herein. The DsiRNAsequences and their target mRNAs are indicated and were manufactured byIDT (Coralville, Iowa). The anti-Renilla siRNA and Ctr-siRNA werepurchased from ABI/Ambion. The sequences provided are SEQ ID NO: 62, SEQID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, from top to bottom.

FIG. 36 provides a comparison of U1 Adaptor with siRNA and ASO Methods.

FIG. 37A provides sequences of the anti-Grm1 Adaptors (top to bottom:SEQ ID NOs: 77-79). All 33 nts are 2′O-methyl RNA. The underlinednucleotides indicate these positions have PS bonds as explained in thetext. The position number indicates in nucleotides where the U1 Adaptoris targeting in the terminal exon of the human Grm1 gene. The terminalexon corresponds to nucleotides 3131-6854 of GenBank Accession No.NM_(—)000838, for example 852 means 852 nts into the terminal exon andwould be nt 3982 of GenBank Accession No. NM_(—)000838. FIG. 37Bprovides a Western blot of transfected C8161 cultured cells probed withan anti-GRM1 antibody (SDIX, Newark, Del.) or a tubulin antibody as aloading control. GAPDH was also probed and shows equal loading. 1million cells were loaded per lane. Transfections were 60 hours. TheWestern blot was repeated three independent times with similar results.The transfection was also repeated 3 independent times with similarresults. FIG. 37C provides a schematic of the predicted structure of theRGD-Vehicle. FIG. 37D provides a graph of tumor suppression observed inmouse C8161 xenografts. FIG. 37E provides a Western blot of proteinlysates from tumors from the FIG. 37D mice. Probing is as in FIG. 37B.

FIG. 38A provides sequences of the anti-BCL2#11 Adaptors and control U1Adaptors (top to bottom: SEQ ID NOs: 80-82). All 33 nts are 2′O-methylRNA. The BCL2#11 target site begins 5 nts into the terminal exon of thehuman BCL2 gene GenBank Accession No. NM_(—)000633 (terminal exon spans1078-6492). FIG. 38B provides a graph of the tumor suppression observedin mouse C8161 xenografts. FIG. 38C provides a graph of tumorsuppression observed in mouse C8161 xenografts.

FIG. 39A provides a representative example of immunohistochemistry oftumor samples from the FIG. 38C mice. The upper row of panels isimmunostained for caspase-3, an indicator of apoptosis, while the lowerrow is immunostained for Ki67, an indicator of proliferation. Thepercentage values indicate the percent of cells that stained positive.FIG. 39 provides a graphic representation of the number ofcaspase-3-positive cells as determined by immunostaining. 10 fields werecounted with the number of positive cells per field indicated.

FIG. 40 shows the tumor suppression observed in mouse C8161 xenograftswith BCL2#12 Adaptor compared to the BCL2#11 Adaptor. Also includes arevariants of the BCL2#11 Adaptor that contains five locked nucleic acid(LNA)-modified nucleotides (top to bottom: SEQ ID NOs: 83-85).

FIG. 41 shows the tumor suppression observed in mouse UACC903 xenograftswith the BCL2#11 Adaptor and a PS-modified variant of BCL2#11 (top tobottom: SEQ ID NOs: 86-87). UACC903-derived tumors typically grow moreslowly than C8161 hence the treatments were of a longer duration.

FIG. 42 provides a schematic for the conjugation of linker and targetingmoiety to an adaptor molecule.

FIGS. 43A and 43B provide images of 8 M Urea 8% PAGE analyses of cRGDmonomer conjugated to atBCL2-A Adaptor and atGRM1-A Adaptor,respectively.

FIG. 44 provides a schematic to conjugate cRGDd to s₂BCL2-A.

FIG. 45 provides an image of an 8 M Urea 8% PAGE analysis of cRGD dimerconjugated to s₂BCL2-A Adaptor with SMCC linker. As seen in lane 5, 40×excess gives a higher yield of conjugation.

FIG. 46A provides an image of an 8 M Urea 8% PAGE analyses of cRGD dimerconjugated to s₂BCL2-A Adaptor with SMCC linker showing that the bestratio of cRGD dimer to LC-SMCC was 1:1.5. FIG. 46B provides an image ofan 8 M Urea 8% PAGE analyses of cRGD dimer conjugated to s₂BCL2-AAdaptor with SMCC linker as a 20× scale up to FIG. 46A.

FIG. 47 provides an image of an 8 M Urea 8% PAGE analysis of theconjugation of cRGD-PEG to s₂BCL2-A. FIG. 47 also provides a schematicof the final product.

FIG. 48 provides graphs showing cRGD-PEG-s₂BCL2-A is active in cellculture to reduce the expression of Bcl2. cRGD-PEG-s₂BCL2-A was activewhen transfected without LF2000 at a higher concentration (100-300 nM)whereas plain BCL2-A was not.

FIG. 49 provides a graph of mean tumor volume for C8161 xenograft micetreated 2×/week by tail vein injection of cRGD-PEG-s₂BCL2-A, vehiclecontrol, or positive control RGD-PPIG5 with 1.7 μg BCL2-A.

FIG. 50 shows a chromatogram of the purification of cRGD-PEG-s₂BCL2-A(0.3 mg scale). FIG. 50 also provides an image of an 8 M Urea 8% PAGEanalysis of Peaks A-E.

FIG. 51 shows a chromatogram of the purification of cRGD-PEG-s₂BCL2-A(1.4 mg scale). FIG. 51 also provides an image of an 8 M Urea 8% PAGEanalysis of Peaks A-E.

FIG. 52 provides an image of an 8% SDS-PAGE analysis of the conjugationof trastuzumab to s₁BCL2-A. Betamercaptoethanol (BetaMe) breaks the S—Sbonds giving a better visualization of the conjugation products.

FIG. 53 provides a graph of BCL2 mRNA after delivery of C1/BCL2-A U1Adaptor/Aptamer or various controls. A549 human lung cells weretransfected 24 hours with U1 Adaptors either with 150 nM PAMAM-G5vehicle (V) or no vehicle “no V”. Y axis is qPCR BCL2 normalized toHPRT1.

FIG. 54 provides images showing that a Cy3-labeled U1 Adaptor localizesto nuclei two hours post-stereotaxic injection into mouse brain(striatum). Upper panels are low magnification, lower panels are highmagnification. Lower panels also show XYZ axis.

FIG. 55 provides a graph of the relative expression of KRAS asdetermined by qPCR analysis in MIA-PaCa2 cells treated with theindicated anti-KRAS U1 Adaptor, anti-KRAS siRNA, or indicated controls.

FIG. 56 provides a graph of xenograft tumor volume after treatment for 8days or 34 days with cRGD-dendrimer vehicle controls or cRGD-dendrimerscomprising KRAS-2 U1 Adaptor, KRAS-3 U1 Adaptor, or BCL2-A U1 Adaptor.

FIG. 57 provides a graph of xenograft tumor volume after treatment withBCL2-A U1 Adaptor either in a cRGD-dendrimer vehicle or without adendrimer as a peptide-Adaptor conjugate.

FIG. 58 provides a graph of xenograft tumor volume after treatment withvehicle control, cRGD-KRAS-3-U1 Adaptor conjugate, or iRGD-KRAS-3-U1Adaptor conjugate.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are methods and compositions for the modulation of theexpression of a gene of interest. The methods comprise the use of the U1adaptor molecule (see, generally, FIG. 1). In its simplest form, the U1adaptor molecule is an oligonucleotide with two domains: (1) anannealing domain designed to base pair to the target gene's pre-mRNA(e.g., in the terminal exon) and (2) an effector domain (also referredto as the U1 domain) that inhibits 3′-end formation of the targetpre-mRNA via binding endogenous U1 snRNP. Without being bound by theory,the U1 adaptor tethers endogenous U1 snRNP to a gene-specific pre-mRNAand the resulting complex blocks proper 3′ end formation. Notably, U1snRNP is highly abundant (1 million/mammalian cell nucleus) and instoichiometric excess compared to other spliceosome components.Therefore, there should be no deleterious effects of titrating outendogenous U1 snRNP.

Preferably, the overall U1 adaptor molecule is resistant to nucleasesand is able to enter cells either alone or in complex with deliveryreagents (e.g., lipid-based transfection reagents). The U1 adaptor oligoshould also be capable of entering the nucleus to bind to pre-mRNA. Thisproperty has already been established in those antisense approaches thatutilize the Rnase H pathway where the oligo enters the nucleus and bindsto pre-mRNA. Additionally, it has been showed that antisense oligos canbind to nuclear pre-mRNA and sterically block access of splicing factorsleading to altered splicing patterns (Ittig et al. (2004) Nuc. AcidsRes., 32:346-53).

The annealing domain of the U1 adaptor molecule is preferably designedto have high affinity and specificity to the target site on the targetpre-mRNA (e.g., to the exclusion of other pre-mRNAs). In a preferredembodiment, a balance should be achieved between having the annealingdomain too short, as this will jeopardize affinity, or too long, as thiswill promote “off-target” effects or alter other cellular pathways.Furthermore, the annealing domain should not interfere with the functionof the effector domain (for example, by base pairing and hairpinformation). The U1 adaptor annealing domain does not have an absoluterequirement on length. However, the annealing domain will typically befrom about 10 to about 50 nucleotides in length, more typically fromabout 10 to about 30 nucleotides or about 10 to about 20 nucleotides. Ina particular embodiment, the annealing domain is at least about 13 or 15nucleotides in length. The annealing domain may be at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 97%, or,more preferably, 100% complementary to the gene of interest. In oneembodiment, the annealing domain hybridizes with a target site withinthe 3′ terminal exon, which includes the terminal coding region and the3′UTR and polyadenylation signal sequences (e.g., through thepolyadenylation site). In another embodiment, the target sequence iswithin about 500 basepair, about 250 basepair, about 100 basepair, orabout 50 bp of the poly(A) signal sequence.

In a particular embodiment, the U1 adaptor may comprise at least onenucleotide analog. The nucleotide analogs may be used to increaseannealing affinity, specificity, bioavailability in the cell andorganism, cellular and/or nuclear transport, stability, and/orresistance to degradation. For example, it has been well-establishedthat inclusion of Locked Nucleic Acid (LNA) bases within anoligonucleotide increases the affinity and specificity of annealing ofthe oligonucleotide to its target site (Kauppinen et al. (2005) DrugDiscov. Today Tech., 2:287-290; Orum et al. (2004) Letters Peptide Sci.,10:325-334). Unlike RNAi and RNase H-based silencing technologies, U1adaptor inhibition does not involve enzymatic activity. As such, thereis significantly greater flexibility in the permissible nucleotideanalogs that can be employed in the U1 adaptor analogs when comparedwith oligos for RNAi and RNase H-based silencing technologies.

Nucleotide analogs include, without limitation, nucleotides withphosphate modifications comprising one or more phosphorothioate,phosphorodithioate, phosphodiester, methyl phosphonate, phosphoramidate,methylphosphonate, phosphotriester, phosphoroaridate, morpholino,amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate,sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilylsubstitutions (see, e.g., Hunziker and Leumann (1995) Nucleic AcidAnalogues: Synthesis and Properties, in Modern Synthetic Methods, VCH,331-417; Mesmaeker et al. (1994) Novel Backbone Replacements forOligonucleotides, in Carbohydrate Modifications in Antisense Research,ACS, 24-39); nucleotides with modified sugars (see, e.g., U.S. PatentApplication Publication No. 2005/0118605) and sugar modifications suchas 2′-O-methyl(2′-O-methylnucleotides) and 2′-O-methyloxyethoxy; andnucleotide mimetics such as, without limitation, peptide nucleic acids(PNA), morpholino nucleic acids, cyclohexenyl nucleic acids,anhydrohexitol nucleic acids, glycol nucleic acid, threose nucleic acid,and locked nucleic acids (LNA) (see, e.g., U.S. Patent ApplicationPublication No. 2005/0118605). See also U.S. Pat. Nos. 5,886,165;6,140,482; 5,693,773; 5,856,462; 5,973,136; 5,929,226; 6,194,598;6,172,209; 6,175,004; 6,166,197; 6,166,188; 6,160,152; 6,160,109;6,153,737; 6,147,200; 6,146,829; 6,127,533; and 6,124,445.

In a particular embodiment, the U1 domain of the U1 adaptor binds withhigh affinity to U1 snRNP. The U1 domain may hybridize with U1 snRNA(particularly the 5′-end and more specifically nucleotides 2-11) undermoderate stringency conditions, preferably under high stringencyconditions, and more preferably under very high stringency conditions.In another embodiment, the U1 domain is perfectly complementary tonucleotides 2-11 of endogenous U1 snRNA. Therefore, the U1 domain maycomprise the sequence 5′-CAGGUAAGUA-3′ (SEQ ID NO: 1). In anotherembodiment, the U1 domain is at least 70%, at least 75%, at least 80%,at least 85%, and more preferably at least 90%, at least 95%, or atleast 97% homologous to SEQ ID NO: 1. The U1 domain may compriseadditional nucleotides 5′ or 3′ to SEQ ID NO: 1. For example, the U1domain may comprise at least 1, 2, 3, 4, 5, or up to 10 or 20nucleotides 5′ or 3′ to SEQ ID NO: 1. Indeed, as demonstratedhereinbelow, increasing the length of the U1 domain to includebasepairing into stem 1 and/or basepairing to position 1 of U1 snRNAimproves the U1 adaptor's affinity to U1 snRNP. The effector domain maybe from about 8 nucleotides to about 30 nucleotides, from about 10nucleotides to about 20 nucleotides, or from about 10 to about 15nucleotides in length. For example, the effector domain may be 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length.

The insertion of point mutations into the U1 domain, i.e., divergingfrom the consensus sequence SEQ ID NO: 1, can moderate silencing.Indeed, altering the consensus sequence will produce U1 domains ofdifferent strength and affinity for the U1 snRNA, thereby leading todifferent levels of silencing. Therefore, once an annealing domain hasbeen determined for a gene of interest, different U1 domains ofdifferent strength can be attached to the annealing domain to effectdifferent levels of silencing of the gene of interest. For examplegAGGUAAGUA (SEQ ID NO: 3) would bind more weakly to U1 snRNP than SEQ IDNO: 1 and, therefore, would produce a lower level of silencing. Asdiscussed above, nucleotide analogues can be included in the U1 domainto increase the affinity to endogenous U1 snRNP. The addition ofnucleotide analogs may not be considered a point mutation if thenucleotide analog binds the same nucleotide as the replaced nucleotide.

Notably, care should be taken so as to not design a U1 adaptor whereinthe effector domain has significant affinity for the target site of themRNA or the sites immediately flanking the target site. In other words,the target site should be selected so as to minimize the base pairingpotential of the effector domain with the target pre-mRNA, especiallythe portion flanking upstream of the annealing site.

To increase the silencing ability of the U1 adaptors, the U1 adaptorshould also be designed to have low self annealing so as to prevent theformation of hairpins within a single U1 adaptor and/or the formation ofhomodimers or homopolymers between two or more U1 adaptors.

The annealing and effector domains of the U1 adaptor may be linked suchthat the effector domain is at the 5′ end and/or 3′ end of the annealingdomain. Further, the annealing and effector domains may be operablylinked via a linker domain. The linker domain may comprise 1, 2, 3, 4,5, 6, 7, 8, 9, 10, up to 15, up to 20, or up to 25 nucleotides.

In another embodiment of the instant invention, more than one U1 adaptordirected to a gene of interest may be used to modulate expression. Asshown hereinbelow, multiple U1 adaptors targeting (annealing) todifferent sequences in the same pre-mRNA should give enhanced inhibition(as has already been shown in FIG. 9). Compositions of the instantinvention may comprise more than one U1 adaptor directed to a particulargene of interest.

In still another embodiment, the U1 adaptor can be combined with othermethods of modulating the expression of a gene of interest. For example,a U1 adaptor can be used in coordination with antisense approaches(Kurreck, J. (2003) Eur. J. Biochem. 270:1628-1644; Bauman et al. (2009)Oligonucleotides 19:1-13) such as, RNase H-based methods, RNAi, miRNA(Lennox et al. (2011) Gene Ther., 18:1111-1120), and morpholino-basedmethods to give enhanced inhibition. Inasmuch as U1 adaptors utilize adifferent mechanism than antisense approaches, the combined use willresult in an increased inhibition of gene expression compared to the useof a single inhibitory agent alone. Indeed, U1 adaptors may target thebiosynthetic step in the nucleus whereas RNAi and certain antisenseapproaches generally target cytoplasmic stability or translatability ofa pre-existing pool of mRNA.

The U1 adaptors of the instant invention may be administered to a cellor organism via an expression vector. For example, a U1 adaptor can beexpressed from a vector such as a plasmid or a virus. Expression of suchshort RNAs from a plasmid or virus has become routine and can be easilyadapted to express a U1 adaptor.

In another aspect of the instant invention, the effector domain of theU1 adaptor can be replaced with the binding site for any one of a numberof nuclear factors that regulate gene expression. For example, thebinding site for polypyrimidine tract binding protein (PTB) is short andPTB is known to inhibit poly(A) sites. Thus, replacing the effectordomain with a high affinity PTB binding site would also silenceexpression of the target gene.

There are U1 snRNA genes that vary in sequence from the canonical U1snRNA described hereinabove. Collectively, these U1 snRNA genes can becalled the U1 variant genes. Some U1 variant genes are described inGenBank Accession Nos. L78810, AC025268, AC025264 and AL592207 and inKyriakopoulou et al. (RNA (2006) 12:1603-11), which identified close to200 potential U1 snRNA-like genes in the human genome. Since some ofthese U1 variants have a 5′ end sequence different than canonical U1snRNA, one plausible function is to recognize alternative splice signalsduring pre-mRNA splicing. Accordingly, the U1 domain of the U1 adaptorsof the instant invention may be designed to hybridize with the 5′ end ofthe U1 variant snRNA in the same way as the U1 domain was designed tohybridize with the canonical U1 snRNA as described herein. The U1adaptors which hybridize to the U1 variants may then be used to modulatethe expression of a gene of interest.

There are many advantages of the U1 adaptor technology to other existingsilencing technologies. Certain of these advantages are as follows.First, the U1 adaptor separates into two independent domains: (1) theannealing (i.e., targeting) activity and (2) the inhibitory activity,thereby allowing one to optimize annealing without affecting theinhibitory activity or vice versa. Second, as compared to othertechnologies, usage of two adaptors to target the same gene givesadditive even synergistic inhibition. Third, the U1 adaptor has a novelinhibitory mechanism. Therefore, it should be compatible when used incombination with other methods. Fourth, the U1 adaptor inhibits thebiosynthesis of mRNA by inhibiting the critical, nearly-universal,pre-mRNA maturation step of poly(A) tail addition (also called 3′ endprocessing).

Although U1in has been successfully used in certain circumstances, itsdevelopment as a widely-used technology has been limited for a varietyof reasons. Certain of these reasons are described as follows.

First, for U1in, there is a possibility that off-target silencing willoccur because a 10 nucleotide sequence, even if it is restricted to theterminal exon, may not be long enough to be unique in the humantranscriptome or in most vertebrate transcriptomes. The U1 adaptorannealing domain does not have restrictions in length or nucleotidecomposition (e.g., nucleotide analogues may be used) and so a length of15 nucleotides, such as was used for LNA6 described hereinbelow, issufficient for uniqueness in a typical mammalian transcriptome.

Second, for U1in, inhibition is readily negated if the pre-mRNA targetsequence is “buried” within intramolecular RNA secondary structure.Indeed, if just half of the 10 nucleotide target (i.e., 5 nucleotides)is base paired, then that is sufficient to block binding of the5′-end-mutated U1 snRNP as well as endogenous U1 snRNP (Fortes et al.(2003) Proc. Natl. Acad. Sci., 100:8264-8269; Abad et al. (2008) NucleicAcids Res. (2008) 36: 2338-2352). This important problem of targetaccessibility is not easily solved and is due in large part to thewell-recognized difficulty algorithms have in accurately predictingshort mRNA secondary structures. The U1 adaptor effector domain is notmasked by RNA structure because it is designed to not base pair with thetarget pre-mRNA.

Third, for U1in, the 5′-end-mutated U1 snRNA is too long to synthesizeas an oligonucleotide and attempts to shorten it while maintainingactivity have failed. Thus, 5′-end-mutated U1 snRNA can only beexpressed from DNA (e.g., from a plasmid or viral delivery system) thathas a suitable U1 snRNA expression cassette. The U1 adaptor is anoligonucleotide that does not have a length restriction and typically isin the range of 20-30 nucleotides in length.

Fourth, for U1in, inhibition by transient transfection of a5′-end-mutated U1 snRNA plasmid is often inefficient because U1 snRNAmaturation takes up to 18 hours, thereby resulting in a significantdelay or “lag time” in accumulation of the inhibitory complex, leadingto a delay in inhibition of the target gene. The U1 adaptor does nothave such a lag time.

Fifth, for U1in, an additional potential concern is that “off-target”effects could arise from basepairing of a 5′-end-mutated U1 snRNP to aninternal exon or intron that then alters the splicing pattern or affectsother steps in the life of that gene's mRNA. This is exacerbated by thefact that the 10 nucleotide targeting sequence for U1in is so short.This concern is mitigated for the U1 adaptor because it has a muchlonger targeting sequence that can also be readily altered.

Sixth, the expression levels of the 5′ end mutated U1 snRNA (theinhibitory molecule for U1in) are significantly lower than the level ofendogenous U1 snRNA (1 million molecules/nucleus) which is thecorresponding inhibitory molecule for the U1 adaptor technology. Thus,inhibitory levels of the U1 adaptor should be higher.

DEFINITIONS

The singular forms “a,” “an,” and “the” include plural referents unlessthe context clearly dictates otherwise.

“Nucleic acid” or a “nucleic acid molecule” as used herein refers to anyDNA or RNA molecule, either single or double stranded and, if singlestranded, the molecule of its complementary sequence in either linear orcircular form. In discussing nucleic acid molecules, a sequence orstructure of a particular nucleic acid molecule may be described hereinaccording to the normal convention of providing the sequence in the 5′to 3′ direction. With reference to nucleic acids of the invention, theterm “isolated nucleic acid” is sometimes used. This term, when appliedto DNA, refers to a DNA molecule that is separated from sequences withwhich it is immediately contiguous in the naturally occurring genome ofthe organism in which it originated. For example, an “isolated nucleicacid” may comprise a DNA molecule inserted into a vector, such as aplasmid or virus vector, or integrated into the genomic DNA of aprokaryotic or eukaryotic cell or host organism.

When applied to RNA, the term “isolated nucleic acid” may refer to anRNA molecule encoded by an isolated DNA molecule as defined above.Alternatively, the term may refer to an RNA molecule that has beensufficiently separated from other nucleic acids with which it would beassociated in its natural state (i.e., in cells or tissues). An isolatednucleic acid (either DNA or RNA) may further represent a moleculeproduced directly by biological or synthetic means and separated fromother components present during its production.

With respect to single stranded nucleic acids, particularlyoligonucleotides, the term “specifically hybridizing” refers to theassociation between two single-stranded nucleotide molecules ofsufficiently complementary sequence to permit such hybridization underpre-determined conditions generally used in the art (sometimes termed“substantially complementary”). In particular, the term refers tohybridization of an oligonucleotide with a substantially complementarysequence contained within a single-stranded DNA molecule of theinvention, to the substantial exclusion of hybridization of theoligonucleotide with single-stranded nucleic acids of non-complementarysequence. Appropriate conditions enabling specific hybridization ofsingle stranded nucleic acid molecules of varying complementarity arewell known in the art.

For instance, one common formula for calculating the stringencyconditions required to achieve hybridization between nucleic acidmolecules of a specified sequence homology is set forth below (Sambrooket al., 1989):

Tm=81.5° C.+16.6 Log [Na+]+0.41(% G+C)−0.63 (% formamide)−600/#bp induplex

As an illustration of the above formula, using [Na+]=[0.368] and 50%formamide, with GC content of 42% and an average probe size of 200bases, the Tm is 57° C. The Tm of a DNA duplex decreases by 1-1.5° C.with every 1% decrease in homology. Thus, targets with greater thanabout 75% sequence identity would be observed using a hybridizationtemperature of 42° C.

The stringency of the hybridization and wash depend primarily on thesalt concentration and temperature of the solutions. In general, tomaximize the rate of annealing of the oligonucleotide with its target,the hybridization is usually carried out at salt and temperatureconditions that are 20-25° C. below the calculated Tm of the hybrid.Wash conditions should be as stringent as possible for the degree ofidentity of the probe for the target. In general, wash conditions areselected to be approximately 12-20° C. below the Tm of the hybrid. Inregards to the nucleic acids of the current invention, a moderatestringency hybridization is defined as hybridization in 6×SSC,5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNAat 42° C., and washed in 2×SSC and 0.5% SDS at 55° C. for 15 minutes. Ahigh stringency hybridization is defined as hybridization in 6×SSC,5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNAat 42° C., and washed in 1×SSC and 0.5% SDS at 65° C. for 15 minutes. Avery high stringency hybridization is defined as hybridization in 6×SSC,5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNAat 42° C., and washed in 0.1×SSC and 0.5% SDS at 65° C. for 15 minutes.

The term “primer” as used herein refers to a DNA oligonucleotide, eithersingle stranded or double stranded, either derived from a biologicalsystem, generated by restriction enzyme digestion, or producedsynthetically which, when placed in the proper environment, is able tofunctionally act as an initiator of template-dependent nucleic acidsynthesis. When presented with an appropriate nucleic acid template,suitable nucleoside triphosphate precursors of nucleic acids, apolymerase enzyme, suitable cofactors and conditions such as a suitabletemperature and pH, the primer may be extended at its 3′ terminus by theaddition of nucleotides by the action of a polymerase or similaractivity to yield a primer extension product. The primer may vary inlength depending on the particular conditions and requirement of theapplication. For example, in diagnostic applications, theoligonucleotide primer is typically 15-25 or more nucleotides in length.The primer must be of sufficient complementarity to the desired templateto prime the synthesis of the desired extension product, that is, to beable anneal with the desired template strand in a manner sufficient toprovide the 3′ hydroxyl moiety of the primer in appropriatejuxtaposition for use in the initiation of synthesis by a polymerase orsimilar enzyme. It is not required that the primer sequence represent anexact complement of the desired template. For example, a noncomplementary nucleotide sequence may be attached to the 5′ end of anotherwise complementary primer. Alternatively, non complementary basesmay be interspersed within the oligonucleotide primer sequence, providedthat the primer sequence has sufficient complementarity with thesequence of the desired template strand to functionally provide atemplate primer complex for the synthesis of the extension product.

Polymerase chain reaction (PCR) has been described in U.S. Pat. Nos.4,683,195, 4,800,195, and 4,965,188, the entire disclosures of which areincorporated by reference herein.

The terms “percent similarity”, “percent identity” and “percenthomology”, when referring to a particular sequence, are used as setforth in the University of Wisconsin GCG software program.

A “replicon” is any genetic element, for example, a plasmid, cosmid,bacmid, phage or virus, which is capable of replication largely underits own control. A replicon may be either RNA or DNA and may be singleor double stranded.

A “vector” is a genetic element, such as a plasmid, cosmid, bacmid,phage or virus, to which another genetic sequence or element (either DNAor RNA) may be attached. The vector may be a replicon so as to bringabout the replication of the attached sequence or element.

An “expression operon” refers to a nucleic acid segment that may possesstranscriptional and translational control sequences, such as promoters,enhancers, translational start signals (e.g., ATG or AUG codons),polyadenylation signals, terminators, and the like, and which facilitatethe expression of a nucleic acid or a polypeptide coding sequence in ahost cell or organism. An “expression vector” is a vector whichfacilitates the expression of a nucleic acid or a polypeptide codingsequence in a host cell or organism.

The term “oligonucleotide,” as used herein, refers to nucleic acidsequences, primers, and probes of the present invention, and is definedas a nucleic acid molecule comprised of two or more ribo ordeoxyribonucleotides, preferably more than three. The exact size of theoligonucleotide will depend on various factors and on the particularapplication and use of the oligonucleotide.

The phrase “small, interfering RNA (siRNA)” refers to a short (typicallyless than 30 nucleotides long, more typically between about 21 to about25 nucleotides in length) double stranded RNA molecule. Typically, thesiRNA modulates the expression of a gene to which the siRNA is targeted.The term “short hairpin RNA” or “shRNA” refers to an siRNA precursorthat is a single RNA molecule folded into a hairpin structure comprisingan siRNA and a single stranded loop portion of at least one, typically1-10, nucleotide.

The term “RNA interference” or “RNAi” refers generally to asequence-specific or selective process by which a target molecule (e.g.,a target gene, protein or RNA) is downregulated via a double-strandedRNA. The double-stranded RNA structures that typically drive RNAiactivity are siRNAs, shRNAs, microRNAs, and other double-strandedstructures that can be processed to yield a small RNA species thatinhibits expression of a target transcript by RNA interference.

The term “antisense” refers to an oligonucleotide having a sequence thathybridizes to a target sequence in an RNA by Watson-Crick base pairing,to form an RNA:oligonucleotide heteroduplex with the target sequence,typically with an mRNA. The antisense oligonucleotide may have exactsequence complementarity to the target sequence or near complementarity.These antisense oligonucleotides may block or inhibit translation of themRNA, and/or modify the processing of an mRNA to produce a splicevariant of the mRNA. Antisense oligonucleotides are typically betweenabout 5 to about 100 nucleotides in length, more typically, betweenabout 7 and about 50 nucleotides in length, and even more typicallybetween about 10 nucleotides and about 30 nucleotides in length.

The term “substantially pure” refers to a preparation comprising atleast 50-60% by weight of a given material (e.g., nucleic acid,oligonucleotide, protein, etc.). More preferably, the preparationcomprises at least 75% by weight, and most preferably 90-95% by weightof the given compound. Purity is measured by methods appropriate for thegiven compound (e.g. chromatographic methods, agarose or polyacrylamidegel electrophoresis, HPLC analysis, and the like).

The term “isolated” may refer to a compound or complex that has beensufficiently separated from other compounds with which it wouldnaturally be associated. “Isolated” is not meant to exclude artificialor synthetic mixtures with other compounds or materials, or the presenceof impurities that do not interfere with fundamental activity or ensuingassays, and that may be present, for example, due to incompletepurification, or the addition of stabilizers.

The term “gene” refers to a nucleic acid comprising an open readingframe encoding a polypeptide, including both exon and (optionally)intron sequences. The nucleic acid may also optionally include noncoding sequences such as promoter or enhancer sequences. The term“intron” refers to a DNA sequence present in a given gene that is nottranslated into protein and is generally found between exons.

As used herein, the term “aptamer” refers to a nucleic acid thatspecifically binds to a target, such as a protein, through interactionsother than Watson-Crick base pairing. In a particular embodiment, theaptamer specifically binds to one or more targets (e.g., a protein orprotein complex) to the general exclusion of other molecules in asample. The aptamer may be a nucleic acid such as an RNA, a DNA, amodified nucleic acid, or a mixture thereof. The aptamer may also be anucleic acid in a linear or circular form and may be single stranded ordouble stranded. The aptamer may comprise oligonucleotides that are atleast 5, at least 10, at least 15, at least 20, at least 25, at least30, at least 35, at least 40 or more nucleotides in length. Aptamers maycomprise sequences that are up to 40, up to 60, up to 80, up to 100, upto 150, up to 200 or more nucleotides in length. Aptamers may be fromabout 5 to about 150 nucleotides, from about 10 to about 100nucleotides, or from about 20 to about 75 nucleotides in length. Whileaptamers are discussed herein as nucleic acid molecules (e.g.,oligonucleotides) aptamers, aptamer equivalents may also be used inplace of the nucleic acid aptamers, such as peptide aptamers.

The phrase “operably linked”, as used herein, may refer to a nucleicacid sequence placed into a functional relationship with another nucleicacid sequence. Examples of nucleic acid sequences that may be operablylinked include, without limitation, promoters, transcriptionterminators, enhancers or activators and heterologous genes which whentranscribed and, if appropriate to, translated will produce a functionalproduct such as a protein, ribozyme or RNA molecule.

“Pharmaceutically acceptable” indicates approval by a regulatory agencyof the Federal government or a state government. “Pharmaceuticallyacceptable” agents may be listed in the U.S. Pharmacopeia or othergenerally recognized pharmacopeia for use in animals, and moreparticularly in humans.

A “carrier” refers to, for example, a diluent, preservative,solubilizer, emulsifier, adjuvant, excipient, auxilliary agent orvehicle with which an active agent of the present invention isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water or aqueous saline solutions and aqueous dextroseand glycerol solutions may be employed as carriers. Suitablepharmaceutical carriers are described, for example, in “Remington'sPharmaceutical Sciences” by E.W. Martin.

Chemotherapeutic agents are compounds that exhibit anticancer activityand/or are detrimental to a cell (e.g., a toxin). Suitablechemotherapeutic agents include, but are not limited to: toxins (e.g.,saporin, ricin, abrin, ethidium bromide, diptheria toxin, andPseudomonas exotoxin); taxanes; alkylating agents (e.g., temozolomide,nitrogen mustards such as chlorambucil, cyclophosphamide, isofamide,mechlorethamine, melphalan, and uracil mustard; aziridines such asthiotepa; methanesulphonate esters such as busulfan; nitroso ureas suchas carmustine, lomustine, and streptozocin; platinum complexes (e.g.,cisplatin, carboplatin, tetraplatin, ormaplatin, thioplatin,satraplatin, nedaplatin, oxaliplatin, heptaplatin, iproplatin,transplatin, and lobaplatin); bioreductive alkylators such as mitomycin,procarbazine, dacarbazine and altretamine); DNA strand-breakage agents(e.g., bleomycin); topoisomerase II inhibitors (e.g., amsacrine,menogaril, amonafide, dactinomycin, daunorubicin, N,N-dibenzyldaunomycin, ellipticine, daunomycin, pyrazoloacridine, idarubicin,mitoxantrone, m-AMSA, bisantrene, doxorubicin (adriamycin),deoxydoxorubicin, etoposide (VP-16), etoposide phosphate, oxanthrazole,rubidazone, epirubicin, bleomycin, and teniposide); DNA minor groovebinding agents (e.g., plicamydin); antimetabolites (e.g., folateantagonists such as methotrexate and trimetrexate); pyrimidineantagonists such as fluorouracil, fluorodeoxyuridine, CB3717,azacitidine, cytarabine, and floxuridine; purine antagonists such asmercaptopurine, 6-thioguanine, fludarabine, pentostatin; asparginase;and ribonucleotide reductase inhibitors such as hydroxyurea);anthracyclines; and tubulin interactive agents (e.g., vincristine,vinblastine, and paclitaxel (Taxol®)).

Radiation therapy refers to the use of high-energy radiation fromx-rays, gamma rays, neutrons, protons and other sources to target cancercells. Radiation may be administered externally or it may beadministered using radioactive material given internally. Chemoradiationtherapy combines chemotherapy and radiation therapy.

As used herein, “oncogene” refers to a gene that when it has higher thannormal activity (e.g., over-expressed), induces abnormal tissue growthdue to effects on the biology of a cell, for example on the cell cycleor cell death process. The term “oncogene” encompasses an overexpressedversion of a normal gene in animal cells (the proto-oncogene) that canrelease the cell from normal restraints on growth (either alone or inconcert with other changes), thereby converting a cell into a tumorcell. Examples of human oncogenes include, without limitation: myc, myb,mdm2, PKA-I (protein kinase A type I), Abl, Bcl1, the anti-apoptoticB-cell lymphoma-2 (Bcl-2) family of proteins (Bcl-2, Bcl-XL, Bcl-w,Mcl-1, Bfl1/A-1, and Bcl-B (see, e.g., Kang et al. (Clin. Cancer Res.(2009) 15:1126-32)), Bcl6, Ras, c-Raf kinase, CDC25 phosphatases,cyclins, cyclin dependent kinases (cdks), telomerase, PDGF/sis, erbA,erb-B, ets, fes (fps), fgr, fms, fos, jun, mos, src, proliferating cellnuclear antigen (PCNA), transforming growth factor-beta (TGF-beta),transcription factors nuclear factor kappaB (NF-κB), E2F, HER-2/neu,TGF-alpha, EGFR, TGF-beta, IGFIR, P12, MDM2, c-myb, c-myc, BRCA, Bcl-2,VEGF, MDR, ferritin, transferrin receptor, IRE, HSP27, hst, int1, int2,jun, hit, B-lym, mas, met, mil (raf), mos, neu (ErbB2), ral (mil),Ha-ras, Ki-ras (Kras), N-ras, rel, ros, sis, ski, trk, yes andmetallothionein genes.

An “antibody” or “antibody molecule” is any immunoglobulin, includingantibodies and fragments thereof, that binds to a specific antigen. Asused herein, antibody or antibody molecule contemplates intactimmunoglobulin molecules, immunologically active portions of animmunoglobulin molecule, and fusions of immunologically active portionsof an immunoglobulin molecule. The term includes polyclonal, monoclonal,chimeric, single domain (Dab) and bispecific antibodies. As used herein,antibody or antibody molecule contemplates recombinantly generatedintact immunoglobulin molecules and immunologically active portions ofan immunoglobulin molecule such as, without limitation: Fab, Fab′,F(ab′)2, F(v), scFv, scFv2, and scFv-Fc.

With respect to antibodies, the term “immunologically specific” refersto antibodies that bind to one or more epitopes of a protein or compoundof interest, but which do not substantially recognize and bind othermolecules in a sample containing a mixed population of antigenicbiological molecules.

The term “treat” refers to the ability of the compound to relieve,alleviate, and/or slow the progression of the patient's disease (e.g.,cancer). In other words, the term “treat” refers to inhibiting and/orreversing the progression of a disease, such as cancer. The term “treat”includes the inhibition, suppression, and/or regression of a tumor.

Compositions and Methods

Compositions of the instant invention comprise at least one U1 adaptorof the instant invention and at least one pharmaceutically acceptablecarrier. The compositions may further comprise at least one other agentwhich inhibits the expression of the gene of interest. For example, thecomposition may further comprise at least one siRNA or antisenseoligonucleotide directed against the gene of interest.

The U1 adaptors of the present invention may be administered alone, asnaked polynucleotides, to cells or an organism, including animals andhumans. The U1 adaptor may be administered with an agent which enhancesits uptake by cells. In a particular embodiment, the U1 adaptor may becontained within a liposome, nanoparticle, or polymeric composition(see, e.g., U.S. Pat. Nos. 4,897,355; 4,394,448; 4,235,871; 4,231,877;4,224,179; 4,753,788; 4,673,567; 4,247,411; 4,814,270; 5,567,434;5,552,157; 5,565,213; 5,738,868; 5,795,587; 5,922,859; and 6,077,663,Behr (1994) Bioconjugate Chem. 5:382-389, and Lewis et al. (1996) PNAS93:3176-3181).

In another embodiment, the U1 adaptor may be delivered to a cell oranimal, including humans, in an expression vector such as a plasmid orviral vector. Expression vectors for the expression of RNA moleculespreferably employ a strong promoter which may be constitutive orregulated. Such promoters are well known in the art and include, but arenot limited to, RNA polymerase II promoters, the T7 RNA polymerasepromoter, and the RNA polymerase III promoters U6 and H1 (see, e.g.,Myslinski et al. (2001) Nucl. Acids Res., 29:2502-09). Viral-mediateddelivery includes the use of vectors based on, without limitation,retroviruses, adenoviruses, adeno-associated viruses, vaccinia virus,lentiviruses, polioviruses, and herpesviruses.

The pharmaceutical compositions of the present invention can beadministered by any suitable route, for example, by injection (e.g.,intravenously and intramuscularly), by oral, pulmonary, nasal, rectal,or other modes of administration. The compositions can be administeredfor the treatment of a disease which can be treated through thedownregulation of a gene(s). The compositions may be used in vitro, invivo, and/or ex vivo. With regard to ex vivo use, the U1 adaptors of theinstant invention (or compositions comprising the same) may be deliveredto delivered to cells (e.g., stem cells) and then re-introduced into thesubject.

The compositions, U1 adaptors, and/or vectors of the instant inventionmay also be comprised in a kit.

The instant invention also encompasses methods of treating, inhibiting(slowing or reducing), and/or preventing a disease or disorder (e.g.,cancer) in a subject. In a particular embodiment, the methods comprisethe administration of a therapeutically effective amount of at least onecomposition of the instant invention to a subject (e.g., an animal orhuman) in need thereof. In a particular embodiment, the compositioncomprises at least one U1 adaptor of the instant invention and at leastone pharmaceutically acceptable carrier. In a particular embodiment, theU1 adaptor is directed to a gene whose over-activity or over-expressionis associated with (e.g., the cause of) the disease or disorder,including viral, fungal, or bacterial infections (see, e.g., EuropeanPatent Application EP2239329 for targets). For example, U1 adaptorsdirected against PCSK9 can be used to treat, inhibit, and/or preventhypercholesterolemia (Frank-Kamenetsky et al. (2008) Proc. Natl. Acad.Sci., 105:11915-20). U1 adaptors directed against Fas (CD95), PTP-1B(protein tyrosine phosphatase 1B), or T cell protein tyrosinephosphatase (TCPTP) can be used to treat, inhibit, and/or preventdiabetes (e.g., type-I or type-II) (Jeong et al. (2010) J. ControlRelease 143:88-94; Xu et al. (2005) Biochem. Biophys. Res. Commun.,329:538-43; Xu et al. (2007) Cell Biol. Intl., 31:88-91). U1 adaptorsdirected against TNFα can be used to treat, inhibit, and/or preventinflammatory diseases (e.g., arthritis) (see also, e.g., targets inPonnappa, B. C. (2009) Curr. Opin. Investig. Drugs, 10:418-24). U1adaptors directed against α-synuclein can be used to treat, inhibit,and/or prevent Parkinson's disease (e.g., Lewis et al. (2008) Mol.Neurodegener., 3:19). U1 adaptors directed against amyloid precursorprotein (APP) or β-secretase can be used to treat, inhibit, and/orprevent neurological disease such as Alzheimer's disease (e.g., Singeret al. (2005) Nat. Neurosci., 8:1343-1349; Miller et al. (2004) NucleicAcids Res., 32:661-8).

In a particular embodiment, the U1 adaptor is directed to a cancer gene(e.g., a gene implicated in the cancer to be treated), particularly anoncogene. For example, the cancer may be characterized byover-expression of the cancer gene to be targeted (e.g., an oncogene).

The instant methods may further comprise the administration of at leastone other agent which inhibits the expression of the target cancer gene.For example, the method may further comprise the administration of atleast one siRNA or antisense oligonucleotide directed against the cancergene. The methods may also comprise the administration at least oneother chemotherapeutic agent or therapy (e.g., radiation). In aparticular embodiment, the chemotherapeutic agent is conjugated to theU1 adaptor (e.g., directly or via a linker; e.g., at the 3′ end and/or5′end). The above agents may be administered in separate compositions(e.g., with at least one pharmaceutically acceptable carrier) or in thesame composition. The agents may be administered simultaneously orconsecutively.

The U1 adaptor of the composition can be targeted to any cancer genewhose expression is to be inhibited in order to treat, inhibit, and/orprevent the cancer. Examples of cancer genes to be targeted are providedhereinabove and in European Patent Application EP2239329 (see, e.g.,89-97), Futreal et al. (Nature Rev. (2004) 4:177-183 (see, e.g.,Supplementary Information S1), Hanahan et al. (Cell (2000) 100:57-70),and Cui et al. (Molecular Systems Biology (2007) 3:152). Examples ofcancer genes to be targeted include, without limitation (examples oftypes of cancer, without limitation, to be treated in parentheses): BCL2(melanoma, lung, prostate cancers or Non-Hodgkin lymphoma), GRM1(melanoma), PDGF beta (testicular and lung cancers), Erb-B (breastcancer), Src (colon cancer), CRK (colon and lung cancers), GRB2(squamous cell carcinoma), RAS (pancreatic, colon and lung cancers, andleukemia), MEKK (squamous cell carcinoma, melanoma or leukemia), JNK(pancreatic or breast cancers), RAF (lung cancer or leukemia), Erk1/2(lung cancer), PCNA(p21) (lung cancer), MYB (colon cancer or chronicmyelogenous leukemia), c-MYC (Burkitt's lymphoma or neuroblastoma), JUN(ovarian, prostate or breast cancers), FOS (skin or prostate cancers),Cyclin D (esophageal and colon cancers), VEGF (esophageal and coloncancers), EGFR (breast cancer), Cyclin A (lung and cervical cancers),Cyclin E (lung and breast cancers), WNT-1 (basal cell carcinoma),beta-catenin (adenocarcinoma or hepatocellular carcinoma), c-MET(hepatocellular carcinoma), PKC (breast cancer), NFKB (breast cancer),STAT3 (prostate cancer), survivin (cervical or pancreatic cancers),Her2/Neu (breast cancer), topoisomerase (ovarian and colon cancers),topoisomerase II alpha (breast and colon cancers), p73 (colorectaladenocarcinoma), p21(WAF1/CIP1) (liver cancer), p27(KIP 1) (livercancer), PPM1D (breast cancer), RAS (breast cancer), caveolin I(esophageal squamous cell carcinoma), MIB I (male breast carcinoma),MTAI (ovarian carcinoma), M68 (adenocarcinomas of the esophagus,stomach, colon, and rectum), mutant p53 (gall bladder, pancreatic andlung cancers), mutant DN-p63 (squamous cell carcinoma), mutant pRb (oralsquamous cell carcinoma), mutant APC 1 (colon cancer), mutant BRCA1(breast and ovarian cancers), mutant PTEN (hamartomas, gliomas, andprostate and endometrial cancers), MLL fusions (acute leukemias),BCR/ABL fusion (acute and chronic leukemias), TEL/AML1 fusion (childhoodacute leukemia), EWS/FLI1 fusion (Ewing Sarcoma), TLS/FUS 1 fusion(Myxoid liposarcoma), PAX3/FKHR fusion (Myxoid liposarcoma), andAML1/ETO fusion (acute leukemia).

Cancers that may be treated using the present invention include, but arenot limited to: cancers of the prostate, colorectum, pancreas, cervix,stomach, colon, endometrium, brain, liver, bladder, ovary, gall bladder,testis, head, neck, skin (including melanoma and basal carcinoma),mesothelial lining, white blood cell (including lymphoma and leukemia),esophagus, breast, muscle, connective tissue, lung (including small-celllung carcinoma and non-small-cell carcinoma), adrenal gland, thyroid,kidney, or bone; glioblastoma, mesothelioma, renal cell carcinoma,gastric carcinoma, sarcoma, choriocarcinoma, cutaneous basocellularcarcinoma, and testicular seminoma.

As stated hereinabove, the U1 adaptors of the present invention may beadministered alone (as naked polynucleotides) or may be administeredwith an agent which enhances its uptake by cells. In a particularembodiment, the U1 adaptor may be contained within a delivery vehiclesuch as a micelle, liposome, nanoparticle, or polymeric composition. Ina particular embodiment, the U1 adaptor is complexed with (e.g.,contained within or encapsulated by) a dendrimer, particularly cationicdendrimers such as poly(amido amine) (PAMAM) dendrimers andpolypropyleneimine (PPI) dendrimers (e.g., generation 3, 4, or 5).

In a particular embodiment, the U1 adaptors are targeted to cancercells. In a particular embodiment, the U1 adaptor is covalently linked(e.g., directly or via a linker) to at least one targeting moiety. Thetargeting moiety may be operably linked to the 5′ end, the 3′ end, orboth ends or to internal nucleotides. In a particular embodiment, one ormore targeting moieties are conjugated to one end of the U1 adaptor(e.g., through a single linker). In a particular embodiment, a complexcomprising the U1 adaptor (e.g., a dendrimer, micelle, liposome,nanoparticle, or polymeric composition) is covalently linked (e.g.,directly or via a linker) to at least one targeting moiety.

Generally, the linker is a chemical moiety comprising a covalent bond ora chain of atoms that covalently attaches the targeting moiety to the U1adaptor complex. The linker can be linked to any synthetically feasibleposition of the targeting moiety and the U1 adaptor or complex(vehicle). In a particular embodiment, the linker connects the targetingmoiety and the U1 adaptor or complex via an amine group and/orsulfhydryl/thiol group, particularly a sulfhydryl/thiol group. Forexample, the U1 adaptor may be derivatized (e.g., at the 5′ end) withone or more amino or thio groups. In a particular embodiment the linkeris attached at a position which avoids blocking the targeting moiety orthe activity of the U1 adaptor. Exemplary linkers may comprise at leastone optionally substituted; saturated or unsaturated; linear, branchedor cyclic alkyl group or an optionally substituted aryl group. Thelinker may also be a polypeptide (e.g., from about 1 to about 20 aminoacids or more, or 1 to about 5). The linker may be biodegradable(cleavable (e.g., comprises a disulfide bond)) under physiologicalenvironments or conditions. In a particular embodiment, the linkercomprises polyethylene glycol (PEG) (alone or in combination withanother linker). In a particular embodiment, the linker is a SPDP(N-Succinimidyl 3-(2-pyridyldithio)-propionate) linker such as LC-SPDP(succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate) or a SMCC(succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) linkersuch as LC-SMCC(succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate)). Thelinker may also be non-degradable (non-cleavable) and may be a covalentbond or any other chemical structure which cannot be substantiallycleaved or cleaved at all under physiological environments orconditions.

Targeting moieties of the instant invention preferentially bind tocancer cells or the relevant tissue or organ. In a particularembodiment, the targeting moiety specifically binds to a markerspecifically (only) expressed on cancer cells or a marker up-regulatedon cancer cells compared to normal cells. The targeting moiety mayspecifically bind to a cancer-specific antigen (e.g., CEA(carcinoembryonic antigen) (colon, breast, lung); PSA (prostate specificantigen) (prostate cancer); CA-125 (ovarian cancer); CA 15-3 (breastcancer); CA 19-9 (breast cancer); HER2/neu (breast cancer); α-fetoprotein (testicular cancer, hepatic cancer); β-HCG (human chorionicgonadotropin) (testicular cancer, choriocarcinoma); MUC-1 (breastcancer); Estrogen receptor (breast cancer, uterine cancer); Progesteronereceptor (breast cancer, uterine cancer); and EGFr (epidermal growthfactor receptor) (bladder cancer)). The targeting moiety may be any typeof compound including, without limitation, peptides, proteins,antibodies (e.g., monoclonal antibodies, antibody fragments, antibodymimics, etc.), lipids, glycoproteins, carbohydrates, small molecules,and derivatives and combinations thereof. In a particular embodiment,the targeting moiety is an antibody or antibody fragment immunologicallyspecific for a surface protein on cancer cells or a surface proteinexpressed at higher levels (or greater density) on cancer cells thannormal cells, tissues, or organs. The antibody or antibody fragment maybe a therapeutic antibody (e.g., possessing a therapeutic effectitself). In a particular embodiment, the targeting moiety is a ligand orbinding fragment thereof for a cell surface receptor on cancer cells. Ina particular embodiment, the targeting moiety is an aptamer. In aparticular embodiment, the targeting moiety specifically binds toalpha-5 beta-3 integrin cell surface receptor. In a particularembodiment, the targeting moiety is an RGD peptide or RGD mimic/analog(see, e.g., European Patent Application EP2239329; U.S. PatentApplication Publication NO. 2010/0280098). The RGD peptide may be,without limitation, a cyclic RGD (cRGD) or internalizing RGD (iRGD). TheRGD peptides may also be a monomer or dimer.

The U1 adaptor molecules of the instant invention may further beconjugated to other desirable compounds. For example, the molecules(e.g., conjugates) may be further conjugated (directly or via a linkeras described above) to detectable agents, therapeutics (e.g., monoclonalantibodies, peptides, proteins, inhibitory nucleic acid molecules, smallmolecules, chemotherapeutic agents, etc.), carrier protein, and agentswhich improve bioavailability, stability, and/or absorption (e.g., PEG).The additional compounds may be attached to any synthetically feasibleposition of the U1 adaptor (or conjugate; (e.g., to the U1 Adaptor(e.g., either end) or the targeting moiety). Alternatively, thetargeting moiety and the U1 adaptor are each individually attached toadditional compound (e.g., carrier protein) (as such the additionalcompound can be considered to serve as the linker between the U1 Adaptorand the targeting moiety). In a particular embodiment, the U1 adaptor isconjugated to a targeting moiety (e.g., cancer cell targeting moiety) atone end and a chemotherapeutic agent on the other. Preferentially, theattachment of the additional compounds does not significantly affect theactivity of the U1 Adaptor or the targeting moiety. Detectable agentsmay be any compound or protein which may be assayed for directly orindirectly, particularly directly. Detectable agents include, forexample, chemiluminescent, bioluminescent, and/or fluorescent compoundsor proteins, imaging agent, contrast agent, radionuclides, paramagneticor superparamagnetic ions, isotopes (e.g., radioisotopes (e.g., ³H(tritium) and ¹⁴C) or stable isotopes (e.g., ²H (deuterium), ¹¹C, ¹³C,¹⁷O and ¹⁸O), optical agents, and fluorescence agents.

Carrier proteins include, without limitation, serum albumin (e.g.,bovine, human), ovalbumin, and keyhole limpet hemocyanin (KLH). In aparticular embodiment, the carrier protein is human serum albumin.Carrier proteins (as well as other proteins or peptides) may beconjugated to the U1 Adaptor conjugate at any synthetically feasibleposition. For example, linkers (e.g., LC-SPDP) may be attached to freeamino groups found on lysines of the carrier protein and then the U1Adaptors and targeting moieties (cRGD dimers) may be conjugated to thelinkers. Any unreacted linkers may be inactivated by blocking withcysteine.

The U1 adaptor molecules of the instant invention may be conjugated(e.g., directly or via a linker) to a compound (e.g., antibodies,peptides, proteins, nucleic acid molecules, small molecules, etc.) whichtargets the U1 adaptor to a desired cell type and/or promotes cellularuptake of the U1 adaptor (e.g., a cell penetrating moiety). Thetargeting moiety may be operably linked to the 5′ end, the 3′ end, orboth ends or to internal nucleotides. In a particular embodiment, thetargeting moiety and/or cell penetrating moiety are conjugated to the 5′end and/or 3′ end. In a particular embodiment, the targeting moietyand/or cell penetrating moiety is conjugated to the 5′ end. In aparticular embodiment, the U1 adaptor molecule is conjugated to both atargeting moiety and a cell penetrating moiety. As used herein, the term“cell penetrating agent” or “cell penetrating moiety” refers tocompounds or functional groups which mediate transfer of a compound froman extracellular space to within a cell. In a particular embodiment, theU1 adaptor is conjugated to an aptamer. The aptamer may be targeted to asurface compound or protein (e.g., receptor) of a desired cell type(e.g., the surface compound or protein may be preferentially orexclusively expressed on the surface of the cell type to be targeted).In a particular embodiment, the aptamer is a cell penetrating aptamer(e.g., C1 or Otter (see, e.g., Burke, D. H. (2012) Mol. Ther., 20:251-253)). In a particular embodiment, the U1 adaptor is conjugated to acell penetrating peptide (e.g., Tat peptides, Penetratin, shortamphipathic peptides (e.g., from the Pep- and MPG-families),oligoarginine, oligolysine). In a particular embodiment, the U1 adaptoris conjugated to a small molecule such as biotin (as part of targetingantibodies) or a non-polar fluorescent group (e.g., a cyanine such asCy3 or Cy5) or to other cell penetrating agents.

In a particular embodiment, at least one of the 3′ end and 5′ end of theU1 adaptor comprises a free-SH group.

The U1 adaptors (including the vehicles comprising the same) describedherein will generally be administered to a patient as a pharmaceuticalpreparation. The terms “patient” and “subject”, as used herein, includehumans and animals. These U1 adaptors may be employed therapeutically,under the guidance of a physician. The U1 adaptors (including thevehicles comprising the same) described herein may also be administeredto humans to provide a desired outcome not involving treatment of adisease (for example, without limitation: personal care cosmetics,enhancement of desired functions both mental and physical).

The compositions comprising the U1 adaptors of the instant invention maybe conveniently formulated for administration with any pharmaceuticallyacceptable carrier(s). For example, the U1 adaptors may be formulatedwith an acceptable medium such as water, buffered saline, ethanol,polyol (for example, glycerol, propylene glycol, liquid polyethyleneglycol and the like), dimethyl sulfoxide (DMSO), oils, detergents,suspending agents or suitable mixtures thereof. The concentration of theU1 adaptors in the chosen medium may be varied and the medium may bechosen based on the desired route of administration of thepharmaceutical preparation. Except insofar as any conventional media oragent is incompatible with the U1 adaptors to be administered, its usein the pharmaceutical preparation is contemplated.

The dose and dosage regimen of U1 adaptors according to the inventionthat are suitable for administration to a particular patient may bedetermined by a physician considering the patient's age, sex, weight,general medical condition, and the specific condition for which the U1adaptors is being administered and the severity thereof. The physicianmay also take into account the route of administration, thepharmaceutical carrier, and the U1 adaptors' biological activity.

Selection of a suitable pharmaceutical preparation will also depend uponthe mode of administration chosen. For example, the U1 adaptors of theinvention may be administered by direct injection to a desired site(e.g., tumor). In this instance, a pharmaceutical preparation comprisesthe U1 adaptors dispersed in a medium that is compatible with the siteof injection. U1 adaptors of the instant invention may be administeredby any method. For example, the U1 adaptors of the instant invention canbe administered, without limitation parenterally, subcutaneously,orally, topically, pulmonarily, rectally, vaginally, intravenously,intraperitoneally, intrathecally, intracerbrally, epidurally,intramuscularly, intradermally, or intracarotidly. In a particularembodiment, the method of administration is by direct injection (e.g.,into the tumor or into the area immediately surrounding the tumor).Pharmaceutical preparations for injection are known in the art. Ifinjection is selected as a method for administering the U1 adaptors,steps must be taken to ensure that sufficient amounts of the moleculesor cells reach their target cells to exert a biological effect.

Pharmaceutical compositions containing a U1 adaptor of the presentinvention as the active ingredient in intimate admixture with apharmaceutically acceptable carrier can be prepared according toconventional pharmaceutical compounding techniques. The carrier may takea wide variety of forms depending on the form of preparation desired foradministration, e.g., intravenous, oral, direct injection, intracranial,and intravitreal.

A pharmaceutical preparation of the invention may be formulated indosage unit form for ease of administration and uniformity of dosage.Dosage unit form, as used herein, refers to a physically discrete unitof the pharmaceutical preparation appropriate for the patient undergoingtreatment. Each dosage should contain a quantity of active ingredientcalculated to produce the desired effect in association with theselected pharmaceutical carrier. Procedures for determining theappropriate dosage unit are well known to those skilled in the art.

Dosage units may be proportionately increased or decreased based on theweight of the patient. Appropriate concentrations for alleviation of aparticular pathological condition may be determined by dosageconcentration curve calculations, as known in the art.

In accordance with the present invention, the appropriate dosage unitfor the administration of U1 adaptors may be determined by evaluatingthe toxicity of the molecules or cells in animal models. Variousconcentrations of U1 adaptors in pharmaceutical preparations may beadministered to mice, and the minimal and maximal dosages may bedetermined based on the beneficial results and side effects observed asa result of the treatment. Appropriate dosage unit may also bedetermined by assessing the efficacy of the U1 adaptors treatment incombination with other standard drugs. The dosage units of U1 adaptorsmay be determined individually or in combination with each treatmentaccording to the effect detected.

The pharmaceutical preparation comprising the U1 adaptors may beadministered at appropriate intervals, for example, at least twice a dayor more until the pathological symptoms are reduced or alleviated, afterwhich the dosage may be reduced to a maintenance level. The appropriateinterval in a particular case would normally depend on the condition ofthe patient.

The following examples describe illustrative methods of practicing theinstant invention and are not intended to limit the scope of theinvention in any way.

Example I

The following methods were used in the Examples II-VIII.

Cultured cells (typically HeLa cells) were grown in media as recommendedby ATCC and seeded the day before transfection such that they would beapproximately 50% confluent on the day of transfection. For24-well-plates, mix #1 and #2 were incubated 15 minutes at roomtemperature and then gently mixed together and incubated another 20minutes at room temperature. Mix #1 was made by adding oligos (adaptors,siRNAs, and M13) and reporter plasmids to 50 μl OPTIMEM® media(Invitrogen catalog 51985; Carlsbad, Calif.). Mix #2 was made by adding1.8 μl LIPOFECTAMINE™-2000 (Invitrogen) to 50 μl OPTIMEM® media. Themedia on the cells in the 24 well dish was removed and 400 μl of freshcomplete media was added. Then all of the Mix 1+2 solution(approximately 110 μl) was added to the cells. For 12-well and 6-wellplate transfections, the values listed above were scaled up 2-fold and4-fold, respectively. For luciferase assays, the cells were harvestedafter 24 hours or 48 hours and luciferase measured using the Promegadual luciferase kit (Madison, Wis.) and a Turner BioSystems Luminometer(Sunnyvale, Calif.). For inhibition of endogenous genes, the cells wereharvested after 24 or up to 48 hours and either lysed in SDS buffer forWestern blotting or total RNA made using a Qiagen RNeasy kit (Valencia,Calif.).

Enhanced chemiluminescence (ECL) Western blotting was done as previouslydescribed (Gunderson et al. (1997) Genes and Dev., 11:761-773; Gundersonet al. (1998) Mol. Cell 1:255-264). Anti-GAPDH antibody (1:10000dilution; Chemicon; Temecula, Calif.), a 1:1000 dilution anti-C-raf-1antibody (R1912 from BD Biosciences; San Jose, Ca) and a 1:1000 dilutionfor the anti-PARP antibody (Ab-2 from Oncogene; La Jolla, Calif.) wereused. The secondary anti-mouse and anti-rabbit antibodies were used at a1:5000 dilution and were obtained from Amersham (Piscataway, N.J.) aswas the chemiluminescent reagent. The membrane used was Immobilon-P fromMillipore (Bedford, Mass.) and was treated as per manufacturer'sinstructions.

RNA from transfected cells was isolated using the Rneasy kit fromQiagen. Complimentary DNA was synthesized using 1 μg of RNA, randomhexamers, and Moloney Murine Leukemia Virus (MMLV) reverse transcriptaseas suggested by the manufacturer (Promega). 50 ng of cDNA was analyzedon real-time PCR using a ROTOR-GENE™ 3000 real time rotary analyzer(Corbett Research; Cambridgeshire, United Kingdom) and QuantiTech SYBRGreen PCR kit (Qiagen). Amplification of glyceraldehyde-3-phosphatedehydrogenase (GAPDH) was used as an endogenous control to standardizethe amount of sample added to the reaction. The comparative cyclethreshold (CT) method was used to analyze the data by generatingrelative values of the amount of target cDNA. To obtain relative values,the following arithmetic formula was used: 2^(−ΔΔCT), whereΔCT=difference between the threshold cycles of the target (c-Raf) and anendogenous reference (GAPDH), and −ΔΔCT=difference between ΔCT of thetarget sample and a control (cells treated with M13 oligo).

Example II

To facilitate testing of the U1 adaptor, the dual luciferase reportersystem from Promega was employed where Firefly luciferase was used as aco-transfected control and Renilla luciferase was targeted forinhibition by the U1 adaptor. Plasmid p717B (FIG. 2A) was constructed bytaking a Promega Renilla luciferase plasmid (pRL-SV40) and replacing its3′UTR and poly(A) signal sequences with sequences from the humanMicrotubule Affinity Regulating Kinase (MARK1) 3′UTR and poly(A) signalregion including 146 basepairs past the poly(A) site. The human MARK13′UTR has a naturally occurring wild type (wt) 10 nucleotide U1 sitethat is also found in other MARK1 homologs in other vertebrates. TheMARK1 wt U1 site in p717B is functional for inhibiting expression.Furthermore, the introduction of a 4 nucleotide mutation in the wt U1site, thereby producing plasmid p717ΔB, resulted in an approximate30-fold increase in Renilla expression (see FIG. 2A). p717ΔB was testedas it would allow for the comparison of “trans-inhibition” mediated by aU1 adaptor:U1 snRNP complex with the “cis-inhibition” mediated by theMARK1 wt U1 site:U1 snRNP complex.

To target p717ΔB for inhibition, a U1 adaptor (FIG. 2B) called LNA6 thatcontains a mixture of Locked Nucleic Acid (LNA) nucleotides andphosphoramidate modified bases was used. In theory, any inhibitoryactivity seen with LNA6 could be due to a combination of two or moreactivities, namely: (1) the binding of U1 snRNP and (2) traditionalantisense effects from its annealing domain, thereby having nothing todo with the effector domain. To distinguish between these activities andfacilitate interpretation of the results, a control U1 adaptor calledLNA7 was used that matches LNA6 except it is unable to bind endogenousU1 snRNP because of a mutation in the effector domain. Any inhibitoryactivity seen with LNA7 would arise solely from the action of itsannealing domain (antisense activity). Therefore, any observedinhibitory activity with LNA6 that was greater than that observed withLNA7 could be attributed to the binding of U1 snRNP to the effectordomain. In other words, comparison of the inhibitory activity of LNA6with LNA7 would indicate how much inhibition is due to endogenous U1snRNP binding the effector domain versus inhibition arising solely fromtraditional antisense effects (e.g., inhibition of translation) that aredue to the annealing activity of the annealing domain.

LNA6 was co-transfected with p717ΔB and the control Firefly reporterinto HeLa cells and after 24 hours the cells were harvested andluciferase activity measured as per the manufacturer's protocol.Parallel experiments were done where LNA7 was used in place of LNA6. Tokeep the amount of transfected oligonucleotide constant, an unrelatedprimer oligonucleotide (the M13 DNA oligonucleotide) was added, wherenecessary, so that the final amount of total oligonucleotide was heldconstant at 62 nM. As seen in FIG. 3A, LNA6 gives a dose dependentinhibitory activity and this activity is far higher than that of LNA7.Indeed, testing of higher concentrations of LNA7 (>62 nM) indicated itsinhibitory activity is approximately the same as that of the M13 DNAoligonucleotide. Therefore, it is evident that nearly all of theinhibitory activity of LNA6 is due to the action of the effector domainrather than antisense effects just from the annealing domain.

In FIG. 3B, the inhibitory activity of LNA6 is plotted as a function ofits concentration and this allows for the calculation of IC₅₀ values,i.e., the concentration of oligo needed to achieve 50% inhibition ofexpression. The IC₅₀ value for LNA6 is 6.35 nM. The IC₅₀ values werecalculated from 3 independent transfections that were plotted as afunction of the U1 adaptor concentration (in this case LNA6) and fittedwith a sigmoidal dose-response function using GrapPad Prism software.

Given that the MARK1 3′UTR contains a natural U1 site it was possiblethat MARK1 sequences flanking the LNA6 binding site contribute to LNA6'sinhibitory activity. To test this, the LNA6 binding site was analyzed inisolation by inserting its binding site into pRL-SV40, which has its3′UTR and poly(A) signal sequences derived from SV40, which is unrelatedto MARK1. This plasmid is called pRL-LNA6 (FIG. 3C). The co-transfectionexperiments and analysis above were repeated where pRL-LNA6 wassubstituted for p717ΔB. It was determined that the IC₅₀ value is 6.86 nMwhich is statistically similar to that seen for the p717ΔB plasmid (FIG.3B). This demonstrates that the LNA6 binding site is necessary andsufficient to confer LNA6's inhibitory activity to the reporter plasmid.

In additional examples, the time of transfection was varied from 24 to48 hours and the amount of transfected plasmid was also varied. Similarresults to those presented above were obtained.

For each targeting site, it may be desirable to optimize the U1 adaptordesign in terms of the number and position of LNA bases so as to giveoptimal inhibition while minimizing off target effects.

It has previously been demonstrated that two U1 snRNP binding sites gavesynergistic enhanced inhibition when inserted into the 3′ terminal exonof a reporter gene (Fortes et al. (2003) Proc. Natl. Acad. Sci.,100:8264-8269). It has also been shown that two 5′-end-mutated U1 snRNAsgave synergistic inhibition when targeting a single endogenous gene(Fortes et al. (2003) Proc. Natl. Acad. Sci., 100:8264-8269). Todemonstrate whether U1 adaptors would behave in the same way, a secondLNA6 binding site was inserted into the pRL-LNA6 plasmid to make thepRL-(LNA6)₂ plasmid (FIG. 4A). The analysis described hereinabove wasrepeated and the results indicate multiple U1 adaptors give enhancedinhibition (FIG. 4B). Notably, such increases in inhibition are rarelyseen when targeting one mRNA with two siRNAs (Elbashir et al. (2001)Nature 411:494-8 Novina et al. (2004) Nature 430:161-4).

Example III

The human C-raf-1 gene was selected to test the U1 adaptor on anendogenous gene. It is well known that the accessibility of the targetsequence is often a rate-limiting step in antisense- and siRNA-basedapproaches. This may also be true for U1 adaptors given their annealingdomains have to target pre-mRNA. Given that antisense-based approachesalso target nuclear pre-mRNA (although for Rnase H-mediated degradationand not poly(A) site inhibition), it was reasoned that a successfulantisense oligonucleotide would imply the target pre-mRNA is availablefor annealing and could be targeted with a U1 adaptor at the same or anearby sequence. Monia et al. (Nat Med. (1996) 2:668-75) screened ˜34antisense oligonucleotides to determine which would be best atinhibiting expression of the C-raf-1 kinase gene. Of the 34, only 2 goodinhibitors were found. Both antisense oligonucleotides were in theterminal exon and are, therefore, candidates for targeting with a U1adaptor.

C-raf-1 is a member of the raf family of genes that are downstreameffectors of ras protein function as part of the MAP kinase signalingpathway (GenBank Accession No. NM_(—)002880). Mutations in raf genestransform cells in vitro and are associated with certain tumors. Highexpression of C-raf-1 mRNA and protein is also found in certain tumors.

As diagrammed in FIG. 5A, a sequence in the 3′UTR of the endogenoushuman C-raf-1 gene was targeted with LNA13. Notably, the U1 domaincomposition is different between LNA6 and LNA13 in that the positions ofthe LNAs were changed. This was done in part to avoid intramolecularbasepairing (e.g., hairpin formation) and intermolecular basepairing(e.g., oligomerization) interactions of the LNA13 adaptor as predictedby computational methods using freely available algorithms such as thosefrom IDT Corporation (Iowa) or Exiqon (Denmark).

HeLa cells are known to express C-raf-1 mRNA and quantitative PCR(Q-PCR) conditions were established to measure mRNA levels of C-raf-1,GAPDH, and Actin. Transfection conditions were as described above withthe M13 oligonucleotide being used to bring the final concentration oftotal oligonucleotide to 62 nM. After 24 hours cells were harvested andused to make total RNA. Q-PCR conditions were: 95° melt, 55° anneal, and72° extend for 15 seconds. The primers used were: C-raf-1 forwardprimer=5′-TGTTTCCAGGATGCCTGTT-3′ (SEQ ID NO: 8), C-raf-1 reverseprimer=5′-GGACATTAGGTGTGGATGTCG-3′ (SEQ ID NO: 9), GAPDH forwardprimer=5′-AGCCACATCGCTCAGACAC-3′ (SEQ ID NO: 10), and GAPDH reverseprimer=5′-GCCCAATACGACCAAATCC-3′ (SEQ ID NO: 11).

Q-PCR was performed using a ROTOR-GENE™ 3000 machine (Corbett LifeSciences) with SYBR Green I detection. The data were analyzed with thecomparative CT method (Pfaffl, M. W. (2001) Nuc. Acid Res.,29:2002-2007) that was adapted to quantitate C-raf-1 mRNA relative toGAPDH mRNA. Results are plotted in FIG. 5B and an IC₅₀ of 17.8 nM wasobserved, a value that compares favorably with that of the bestantisense oligonucleotide (out of 34 tested) called “ISIS 5132” in Moniaet al. (Nat Med. (1996) 2:668-75) that had an IC₅₀=50 nM. Notably, LNA13cannot act through the Rnase H cleavage pathway because it contains asufficient number of modified nucleotides so that Rnase H activity isinhibited (Kurreck et al. (2002) Nuc. Acids Res., 30:1911-8). ThusLNA13's IC_(so) value of 17.8 nM does not arise from the Rnase Hcleavage pathway.

The C-raf-1 3′UTR and sequences past the poly(A) site were subclonedinto a Renilla reporter to make the pRL-wtC-raf-1 plasmid. This allowedfor the direct comparison of the IC₅₀ values with other Renilla reporterplasmids as discussed hereinabove. As shown in FIG. 6A, co-transfectionof pRL-wtC-raf-1 with LNA13 gave an IC₅₀ value of 7.98 nM which issimilar to the IC₅₀ value seen with the endogenous C-raf-1 gene. Somedifferences may be expected as the pRL-wtC-raf-1 plasmid produces achimeric mRNA that may behave differently than the endogenous gene.

To determine the intrinsic inhibitory activity of LNA13, a single LNA13binding site was inserted into pRL-SV40 and inhibition was tested asdescribed above. An IC₅₀ value of ˜2 nM was determined. Thus, theinhibitory activity of a single U1 adaptor, in this case LNA13, varieswhen tested against its endogenous target gene, a reporter plasmid withthe natural 3′UTR of the target gene (FIG. 6A), and a reporter plasmidwith the isolated binding site (FIG. 6B). Without being bound by theory,the differences in activity may be due to accessibility. Accessibilityfactors includes (1) folding of the pre-mRNA sequence, (2) binding oftrans-acting factors, and (3) the rate of 3′ end processing of thepre-mRNA.

Example IV

The U1 domain contributes to U1 adaptor activity by its affinity to U1snRNA and more broadly to the U1 snRNP complex. Although the U1 domainsequence is fixed (unless variant U1 snRNAs are targeted), the U1 domainsequence can be lengthened and its composition can be changed. To thisend, replacing the LNA-DNA mixmer design with 100% 2′-O-methyl resultedin only a small decrease in activity (FIG. 7). Notably, 2′-O-methylnucleotides are easier to use during synthesis and have a lower cost ascompared to LNA-DNA mixmers. Additionally, having a uniform U1 domaincomposition simplifies adaptor design as the focus is then on optimizingthe annealing domain. Having 100% 2′-O-methyl also reduces selfannealing problems as compared to having an LNA-DNA mixmer design orother mixmer combinations. The mixmer annealing domain of LNA6 was alsoreplaced with a matching sequence comprising 100% 2′-O-methyl. However,the adaptor comprising only 2′O-methyl showed reduced activity.Extension of the annealing domain (Ome-5 U1 adaptor) failed to restoreactivity indicating that the presence of only 2′O-methyl in theannealing domain could not simply be compensated for by a longerannealing domain.

The U1 adaptor design has the advantage that inhibition does not requireenzymatic activity. Thus, a variety of modified bases may beincorporated into its design. Phosphorothioate (PS) bonds are typicallyincorporated into antisense molecules to improve their stability whendelivered into cells. To test whether PS bonds would affect activity,the activity of two matched adaptors LNA17 and LNA21 that differ only inthat LNA21 has PS bonds were compared. As can be seen in FIG. 8, thesetwo matched adaptors had similar activities, thereby indicating that PSbonds did not effect activity.

To test whether the U1 domain can be moved to the other end of the U1adaptor, a set of matching adaptors where the U1 domain was placed ateither the 5′ or 3′ of the annealing domain was synthesized. Theactivity of these two adaptors was found to be comparable, therebyindicating U1 snRNP access to the U1 domain does not depend on itsposition relative to the annealing domain. A U1 adaptor comprising twoU1 domains on both sides (i.e., a multivalent adaptor) was alsosynthesized. However, no significant change in activity was found ascompared to the monovalent adaptors. This suggests that U1 snRNP bindingis not the limiting factor for inhibitory activity in vivo. Linker baseswere also inserted between the annealing and U1 domains. Notably, noloss or improvement in U1 adaptor activity was found.

Example V

A series of adaptors were synthesized and tested where the U1 domain washeld constant and the length and composition of the annealing domainwere varied. As seen in FIG. 9, shortening the annealing domain can leadto a reduction in adaptor activity. Furthermore, reducing thebasepairing potential by substituting DNA bases for LNAs can also reduceactivity.

The U1 domain length of the U1 adaptors described hereinabove has beenlimited to 10 nucleotides, mostly because its natural consensus bindingsite (i.e., the 5′ss) is 9 to 10 nucleotides long. The 5′-mostnucleotide of U1 snRNA is an A and is not thought to play a role in 5′ssbinding. However, the effect of this nucleotide on U1 adaptor activitywas tested. Matching adaptors that differ only by 1 nucleotide in the U1domain were compared. As shown in FIG. 10, the 1 nucleotide-extended U1domain gives a significant increase in inhibitory activity

Based on the above results with the additional nucleotide, a matchedseries of adaptors (the LNA17 series) that incrementally vary the U1domain length from 7 to 13 nucleotides was tested. U1 adaptor activitywas found to steadily increase from no activity (7 nucleotide U1 domain)to high activity (13 nucleotide U1 domain). Given the adaptors in FIGS.9 and 10 have different annealing domains, it can be concluded thatimprovement by longer U1 domains does not depend on the annealingdomain. Based on the known structure of U1 snRNA, the higher activitymay be because the 12th and 13th nucleotides of the U1 domain insertthemselves into stem 1A of U1 snRNA. Stem 1A is highly conserved in U1snRNAs from yeast to humans, suggesting they are functionally important.Further extensions of the U1 domain will eventually disrupt U1 snRNPconformation, such as disrupting binding of U1-70K to stem loop 1. Thus,further extensions of the adaptors will eventually lead to inactive U1adaptor activity because of disruption of U1 snRNP inhibitory activity.

Example VI

The U1 adaptors were tested in a variety of cell types. It has beenpreviously shown that U1in-based gene silencing is active in a broadvariety of vertebrate cell lines and primary cells (Fortes et al. (2003)Proc. Natl. Acad. Sci., 100: 8264-8269). To test U1 adaptors, theLNA17-11 adaptor was transfected into the cell lines shown in FIG. 12.U1 adaptor activity was found in all cases, though there was somevariance in the amount of inhibition.

Example VII

As discussed hereinabove, U1 adaptors and siRNA utilize distinctmechanisms that occur in different compartments of the cell (nucleusversus cytoplasm). To determine whether their combined usage to silencea single gene would give enhanced inhibition, the pRL-LNA6 Renillareporter plasmid was targeted with 1 nM anti-Renilla siRNA (RL-siRNA)from Ambion/ABI (catalog 4630; Austin, Tex.) and 30 nM LNA6 adaptor.Control siRNA (Ctr-siRNA) from Ambion/ABI (catalog 4611G) and the LNA7control adaptor were used as controls. As shown in FIG. 13A, theco-transfection of RL-siRNA with LNA6 gave markedly enhanced inhibitionof Renilla expression when compared to use of the control oligos.

To rule out that these results depend on the type of adaptor andreporter, an anti-GAPDH U1 adaptor (LNA12) was used in place of LNA6 andthe reporter plasmid having an annealing site for LNA12 because itcontains the 3′UTR of the human GAPDH mRNA (FIG. 13B). Morespecifically, pRL-GAPDH is a Renilla reporter with the 3′UTR and poly(A)signal sequences derived from the human GAPDH mRNA (GenBank AccessionNo. NM_(—)002046) plus 200 basepairs past the GAPDH poly(A) signal. TheLNA12 adaptor is targeting 1231-1245, using Gen Bank Accession No.NM_(—)002046 coordinates.

As shown in FIG. 13C, enhanced activity was observed when 15 nM LNA12and 1 nM RL-siRNA were used together. Although the siRNAs used in theseexperiments are more active in silencing than the U1 adaptors, it shouldbe noted that the siRNAs were optimized over the course of years toproduce such highly active siRNAs to GAPDH and to Renilla reporterplasmids.

In view of the above data, the combination of U1 adaptors with moretraditional antisense-based methods that employ RNase H activity weretested. In this experiment, the pRL-wtC-raf reporter plasmid wastargeted. The LNA25-H/U1 oligo combines into one molecule both adaptorand Rnase H activities by designing the annealing domain to have anuninterrupted stretch of at least seven DNA bases (in this case 10bases) as seen for LNA25-H/U1. Such a “7 nt DNA design” was shown byGrünweller et al. (Nuc. Acids Res. (2003) 31:3185-93) to be sufficientfor Rnase H activity, although longer stretches are more active.LNA25-mtH/U1 matches LNA25-H/U1 but has the stretch of DNAs interruptedand so should not have Rnase H activity. In like manner, LNA25-H/mtU1matches LNA25-H/U1 but has a 2 nucleotide mutation in the U1 domain(FIG. 14A). This design is such that LNA25-H/mtU1 has only Rnase Hactivity, LNA25-mtH/U1 has only U1 adaptor activity, and LNA25-H/U1 willhave both activities. As can be seen in FIG. 14B, the LNA25-H/U1 hashigher activity indicating both silencing methods can give enhancedactivity when used together. Notably, DNA stretches that are longer than10 nucleotides can be used, however the potential for self annealing ofoligos with longer DNA stretches needs to be considered.

Example VIII

To determine whether the LNA25 series of oligonucleotides can inhibitexpression of the endogenous C-raf-1 gene, HeLa cells were transfectedwith LNA25-mtH/U1 and Western blotting combined with Q-PCR wasperformed. The results in FIG. 15 show specific silencing of C-raf-1both at the protein and mRNA levels. It has been reported that silencingof C-raf-1 leads to induction of cleavage of the PARP protein as part ofinduction of apoptosis (Lau et al. (1998) Oncogene 16:1899-902).Re-probing the Western blot in FIG. 15A with anti-PARP antibodydemonstrated that the U1 adaptors induce PARP cleavage indicative ofC-raf-1 silencing.

As described hereinabove, the combinatorial use of adaptors and Rnase Hgave enhanced silencing of a reporter plasmid. To determine whetherenhanced activity could be extended to silencing of the endogenousC-raf-1 gene, the above transfections in Example VII were repeated, butnow the levels of the endogenous C-raf-1 protein and mRNA were measured.The results in FIG. 16 show enhanced silencing of C-raf-1 both at theprotein and mRNA levels.

Example IX Introduction

Use of RNAi to silence specific vertebrate genes has rapidly become astandard method for gene function analysis and has garnered muchattention as a promising new molecular therapy (Hannon et al. (2004)Nature 431:371-8; Kim et al. (2007) Nat. Rev. Genet., 8:173-84). RNAisilences gene expression by degrading the target mRNA in the cytoplasmand typically employs synthetic siRNA duplexes (Elbashir et al. (2001)Nature 411:494-8) or engineered vectors (plasmid or viral) that expresslonger precursor RNAs (e.g., short hairpin shRNAs). An alternative genesilencing method called U1i (U1 small nuclear RNA-U1 snRNA-interference)that uses a plasmid vector to express an engineered U1 snRNA (called aU1i snRNA) in which a 10 nucleotide (nt) sequence complementary to thetarget gene's terminal exon replaces the natural U1 targeting domain waspreviously published (Beckley et al. (2001) Mol. Cell Biol., 21:2815-25;Fortes et al. (2003) Proc. Natl. Acad. Sci. USA 100: 8264-8269). The U1isnRNA assembles into a U1 snRNP complex that basepairs to the targetgene's pre-mRNA and inhibits polyA tail addition, an obligatory RNAprocessing step for nearly all eukaryotic mRNA (Fortes et al. (2003)Proc. Natl. Acad. Sci. USA 100: 8264-8269; Liu et al. (2004) NucleicAcids Res., 32:1512-7). Without polyadenylation, the pre-mRNA fails tomature and is degraded in the nucleus, thereby reducing levels ofcytoplasmic mRNA of the target gene. The mammalian U1 snRNP consists of10 proteins bound to the 164 nt U1 snRNA and functions early in splicingvia a base pairing interaction between U1 snRNA and the 5′ splice sitesequence (Will et al. (1997) Curr. Opin. Cell Biol., 9:320-8). Separatefrom its role in splicing, U1 snRNP can also be a potent inhibitor ofgene expression when it is bound near the polyA signal of the pre-mRNA.This was first shown in papillomaviruses (Furth et al. (1994) Mol. Cell.Biol., 14:5278-5289) and more recently in certain mammalian genes (Guanet al. (2007) RNA J., 13:2129-2140) and it is this property of U1 snRNPthat forms the basis of the U1i silencing method. The inhibitorymechanism involves the U1-70K subunit of U1 snRNP binding to andinhibiting the activity of polyA polymerase (Gunderson et al. (1998)Mol. Cell, 1:255-264).

Although U1i is effective in reducing mRNA levels, it has not beenwidely adopted as a gene silencing technology due to the inconvenienceof preparing custom U1i targeting plasmids and concerns overspecificity. U1i binds the target mRNA using a 10 nt domain engineeredonto the 5′-end of the U1 snRNA. Lengthening this 10 nt domainparadoxically results in weaker silencing. Furthermore, the U1i snRNAmust be expressed off a plasmid or viral vector and attempts to shortenits length while maintaining activity, so as to be amenable to chemicalsynthesis, have not been successful.

These problems are circumvented herein by employing a syntheticoligonucleotide, a U1 Adaptor, to recruit endogenous U1 snRNP to thetarget site. The U1 Adaptor has two domains: a “Target Domain” designedto base pair to the target gene's pre-mRNA in the 3′ terminal exon, anda “U1 Domain” that tethers U1 snRNP to the target. Bringing the U1 snRNPin contact with the target pre-mRNA inhibits proper 3′-end formation andeventually leads to RNA degradation. Using optimized U1 Adaptor designand chemical modifications to improve binding affinity, very highpotency is seen and subnanomolar IC₅₀ (the concentration needed toinhibit gene expression by 50%) can be achieved. Interestingly,targeting the same gene either with multiple U1 Adaptors or byco-transfection of U1 Adaptors with siRNAs gives synergistic higherlevels of inhibition, the latter probably because U1 Adaptors and siRNAsutilize distinct mechanisms and act in distinct cellular compartments.U1 Adaptors add a new dimension to the gene silencing tool kit and canbe used either as a stand-alone method or in combination with RNAi.

Materials and Methods Method for Transfection and Luciferase Assays

Cell culture and transfections were done as previously described(Goraczniak et al. (2008) J. Biol. Chem., 283:2286-96). For luciferaseassays, the cells were harvested after 24-48 hours and luciferasemeasured using the Promega dual luciferase kit (Promega, Madison, Wis.)measured on a Turner BioSystems Luminometer (Turner BioSystems,Sunnyvale, Calif.). For inhibition of endogenous genes, cells wereharvested after 24-48 hours and either lysed in SDS buffer for Westernblotting or total RNA was extracted using the RNeasy kit (Qiagen,Valencia, Calif.). Nuclear and cytoplasmic RNA preparations wereperformed as described (Goraczniak et al. (2008) J. Biol. Chem.,283:2286-96). The anti-Renilla siRNA and was purchased from ABI/Ambion(Austin, Tex.). All of the U1 Adaptors and the anti-PCSK9 siRNA andanti-RAF1 siRNA were manufactured by Integrated DNA Technologies (IDT,Coralville, Iowa). A list of sequences for all the U1 Adaptors andsiRNAs is provided in FIGS. 34 and 35.

ECL Western Blots

ECL Western blotting was done as previously described (Gunderson et al.(1998) Mol. Cell 1:255-264) using a 1:10000 dilution of an anti-GAPDHantibody (Chemicon division of Millipore, Billerica, Mass.), a 1:1000dilution of an anti-RAF1 antibody (R1912 from BD Biosciences, San Jose,Calif.), or a 1:1000 dilution of an anti-PARP antibody (Ab-2 fromOncogene, Cambridge, Mass.). The secondary anti-mouse and anti-rabbitantibodies were used at a 1:5000 dilution (Amersham Biosciences,Piscataway, N.J.). The membrane used was Immobilon-P (Millipore) and wastreated as per manufacturer's instructions.

General Method for Quantitative Real-Time PCR (qPCR)

RNA from transfected cells was isolated using the RNeasy kit (Qiagen).Complementary DNA (cDNA) was synthesized using 1 μg of RNA, randomhexamers and MMLV reverse transcriptase as suggested by the manufacturer(Promega). 50 ng of cDNA was analyzed using qPCR run on a Rotorgene 3000(Corbett Research, Sydney, Australia) and the QuantiTech SYBR Green PCRkit (Qiagen). Results from test genes were normalized using GAPDH as aninternal control. Primer sequences are provided herein. The comparativecycle threshold (Ct) method was used (Pfaffl, M. W. (2001) Nucleic AcidRes., 29:2002-2007) to analyze the data where the relative values of theamount of target cDNA equal 2-ΔΔCt, where ΔCt=difference between thethreshold cycles of the target (RAF1) and an endogenous reference(GAPDH), and −ΔΔCt=difference between ΔCt of the target sample and acontrol (cells treated with M13 oligo).

Preparation and Analysis of RNA

RPA and the uniformly ³²P-labelled RNA probes were made by in vitrotranscription with T7 or SP6 RNA polymerase in the presence of 32P-UTPas described (Goraczniak et al. (2008) J. Biol. Chem., 283:2286-96). TheqPCR conditions were: 95° C. melting temperature, 55° C. annealingtemperature and 72° C. extension temperature each for 15 seconds. Thesequences of the oligonucleotides used to measure GAPDH and cRAF by qPCRare given below (from top to bottom: SEQ ID NOs: 8-11, 66, 67).

C-raf-1 forward primer = 5′-TGTTTCCAGGATGCCTGTT C-raf-1 reverse primer =5′-GGACATTAGGTGTGGATGTCG GAPDH forward primer = 5′-AGCCACATCGCTCAGACACGAPDH reverse primer = 5′-GCCCAATACGACCAAATCC PCSK9 forward primer =5′- ATGTCGACTACATCGAGGAGGACT PCSK9 reverse primer =5′-TGGTCACTCTGTATGCTGGTGTCT

The sequences of the oligonucleotides used for RT-PCR are given below(from top to bottom: SEQ ID NOs: 68-75).

Cdc25B forward primer = 5′-CCATCAGACGCTTCCAGTCT Cdc25B reverse primer =5′-GTCTCTGGGCAAAGGCTTC Cdc25C forward primer = 5′-TGGCTCAGGACCCAGTTTTACdc25C reverse primer = 5′-TCTTCTGCCTGGTCTTCTCC Grb2 forward primer =5′-CGCGAAGCTTGTTTTGAACGAAGAATGTGATCAG Grb2 reverse primer =5′-GAGAGGTACCCTGTGGCACCTGTTCTATGTCCCGCAGGAATATCFibronectin forward primer = 5′-TGCGGTACCGGCCTGGAGTACAATGTCAFibronectin reverse primer = 5′-TGCGGTACCGAGGTGACACGCATGGTGTC

Western Blotting

The anti-PARP antibody employed was Ab-2 from Oncogene (La Jolla,Calif.). The secondary anti-mouse and anti-rabbit antibodies were usedat a 1:5000 dilution (Amersham, Piscataway, N.J.) as was thechemiluminescent reagent. The membrane used was Immobilon-P (Millipore,Bedford, Mass.) and was treated as per the manufacturer's instructions.

EMSA

U1 snRNP was purified as previously described (Abad et al. (2008)Nucleic Acids Res., 36:2338-52; Gunderson et al. (1998) Molecular Cell,1: 255-264). For FIG. 18, gel purified, ³²P-radiolabeled RNA probe wasincubated with purified U1 snRNP and U1 Adaptor as indicated in bindingbuffer (BB) (BB=20 mM HEPES-KOH (pH 7.9), 100 mM KCl, 1.5 mM MgCl2, 5 mMdithiothreitol, 5% glycerol, 15 tRNA) in a total volume of 15 μl at roomtemperature for 15 min. The protein-RNA complexes were then loaded on a6% polyacrylamide gel with 1×TBE and 5% glycerol and electrophoresed for2.5 hours at 20V/cm. Gels were dried and used first for autoradiographyfollowed by phosphoimagery analysis to quantitate the complexes asdescribed (Abad et al. (2008) Nucleic Acids Res., 36:2338-52). For FIG.20, the U1 snRNP was first bound to unlabeled U1 Adaptor and, after 15minutes, the radiolabeled RNA probe was added and 10 minutes later PAGEwas performed as in FIG. 18.

Microarray Analysis

Microarray analysis and data interpretation were performed at the CancerInstitute of New Jersey (CINJ) Microarray facility that offers completeAffymetrix GeneChip technology including data analysis. For each gene,dividing the anti-PCSK9 DsiRNA by the M13 control is a measure of the-fold change of that gene due to the anti-PCSK9 DsiRNA. Likewisedividing the anti-PCSK9 UA31d4+UA31e Adaptors by the M13 control is ameasure of the -fold change of that gene due to the anti-PCSK9 Adaptors.The data were filtered by excluding out both “Absent” genes (due tovery-low-expression) as well as genes where the p values were >0.05 orthat had “zeros” in the change call (i.e. those genes that did notsignificantly change). Out of 54,000 human transcripts represented onthe chip, about 4000 had changes ≧2-fold (that is R2/R1 and R3:R1 were≧2-fold). These 4000 were ranked according to genes with the largestdecrease and it was found that PCSK9 ranked seventh highest for theanti-PCSK9 Adaptors and first for the anti-PCSK9 DsiRNA consistent withit being the target for silencing.

Results U1 Adaptor Oligonucleotides Reduce Gene Expression

To facilitate rapid analysis, the dual luciferase reporter system wasused where Renilla luciferase mRNA was targeted for inhibition by U1Adaptors and a co-transfected Firefly luciferase reporter served as aninternal normalization control. The first target studied was MARK1(NM_(—)018650), which contains a single natural U1 snRNP binding site(U1 site) in its 3′-UTR that downregulates MARK1 expression in thewildtype (wt) gene (Guan et al. (2007) RNA J., 13:2129-2140). Thereporter pRL-MARK1 wt was made from a standard pRL-SV40 Renillaexpression plasmid by replacing the SV40-derived 3′UTR and polyA signalsequences with the human MARK1 3′UTR and polyA signal region, including146 nt past the polyA site. The pRLMARK1mt reporter matches pRL-MARK1 wtexcept for a four base change in the natural U1 site. Each MARK1reporter was transfected into HeLa cells along with a control Fireflyreporter. It was observed that the pRL-MARK1mt plasmid has a 17-foldincrease in Renilla luciferase expression as compared to the pRL-MARK1wt plasmid indicating that the natural U1 site causes a 17-fold level ofinhibition in the wt reporter (see hereinabove). The fact that the wtMARK1 3′UTR can be inhibited by a U1 snRNP-mediated mechanism indicatedthat this sequence context would be a good first test for the U1 Adaptormethod. A 25 nt U1 Adaptor oligonucleotide called UA6 for U1 Adaptor 6,was designed with a 10 nt U1 Domain complementary to the 5′-end of theU1 snRNA and a 15 nt Target Domain complementary to MARK1 sequenceimmediately 3′ to the mutated U1 binding site in pRL-MARK1mt. UA6 hasten locked nucleic acid (LNA) nucleotides with the other positions beingDNA nucleotides. Co-transfection of the UA6 Adaptor with the pRL-MARK1mtplasmid and the control Firefly reporter into HeLa cells resulted in a90% inhibition of Renilla luciferase expression at 62 nM concentrationwith an IC₅₀ of 6.6 nM (see hereinabove). A Ribonuclease ProtectionAnalysis (RPA) with a Renilla mRNA-specific probe (Goraczniak et al.(2008) J. Biol. Chem., 283:2286-96) demonstrated reduction in both totaland cytoplasmic Renilla mRNA levels, indicating that inhibition occursat the RNA level with no apparent nuclear accumulation of the RenillamRNA (FIG. 17). To demonstrate UA6 inhibition requires complementaritywith U1 snRNA, a mismatch control Adaptor, UA7a, that has a 4 nucleotidemutation in the U1 Domain, was synthesized and tested. A 4 out of 10base mismatch in this domain reduces complementarity with U1 snRNA sothat it no longer binds U1 snRNP. Pre-mRNAs containing this same 4 ntmutation are unable to bind U1 snRNP as compared to a matching pre-mRNAwith a wild type U1 Domain sequence using an electrophoretic mobilityshift assay (EMSA) and purified U1 snRNP (Gunderson et al. (1998) Mol.Cell 1:255-264; Abad et al. (2008) Nucleic Acids Res.; 36:2338-52). Asimilar EMSA was used to directly demonstrate that the UA6 Adaptor cantether the U1 snRNP complex to the target RNA (FIG. 18). Co-transfectionof the mutant UA7a Adaptor with pRL-MARK1mt plasmid resulted in noinhibition (see hereinabove), demonstrating the importance of the U1Domain.

The chemical composition and design of the U1 Adaptors is crucial forfunction. All “first generation” U1 Adaptors were LNA/DNA mixmers. LNAnucleotides contain a carbon linkage between the 2′-oxygen and the 4′carbon of the ribose sugar ring thereby “locking” the nucleotide in an“endo-sugar pucker” position leading to higher duplex stability andrelative resistance to nuclease degradation (Kauppinen et al. (2005)Drug Discovery Today: Technol., 2:287-290). LNA nucleotides wereincluded in the U1 Adaptor to increase binding affinity of the shortfunctional domains present in the 25 nt oligonucleotide. Placement ofLNA nucleotides in this pattern also avoids activation of an RNaseH-dependent “antisense” silencing mechanism. 2′-modification of theribose, such as 2′-O-methyl (2′OMe), LNA, or 2′-Fluoro, blocks RNase Hactivity. RNase H activation requires at least 4 contiguous DNA residuesand does not reach full potency until 7-8 DNAs are present (Kurreck etal. (2002) Nucleic Acids Res., 30:1911-8; Grünweller et al. (2003)Nucleic Acids Res., 31:3185-93). The fact that all of the active U1Adaptors in this report have ≦4 continuous DNA nucleotides arguesagainst a role for RNase H in U1 Adaptor activity. U1 Adaptorconfigurations that support both RNase H activity and U1 snRNP bindingin the same molecule may increase potency by exploiting two differentmechanisms of action.

It is possible that MARK1 sequences flanking the UA6 binding sitecontribute to the observed suppression. To rule out this possibility,the 15 nt UA6 binding site was tested outside of the context of theMARK1 3′UTR by construction of a reporter called pRL-UA6 that has oneUA6 binding site inserted into the 3′UTR and polyA signal sequencederived from SV40 (see hereinabove). Co-transfection of pRL-UA6 withincreasing amounts of the UA6 Adaptor suppressed expression of Renillaluciferase with an IC₅₀ value of 7.4 nM, which is nearly identical tothe IC₅₀ of 6.6 nM seen for the UA6 Adaptor against the pRL-MARK1mtreporter. As shown hereinabove, the mutated UA7a Adaptor did not showany inhibitory activity. Thus the 15 nt UA6 binding site is necessaryand sufficient to quantitatively direct inhibition by the UA6 Adaptoroligonucleotide. It has been previously demonstrated that multiple U1snRNP binding sites in the terminal exon show additive levels ofinhibition (Beckley et al. (2001) Mol. Cell Biol., 21:2815-25; Fortes etal. (2003) Proc. Natl. Acad. Sci. USA, 100:8264-8269; Liu et al. (2004)Nucleic Acids Res., 32:1512-7). A new version of the pRL-UA6 reporterwas made that had two tandem UA6 binding sites, called pRL-(UA6)₂. Asshown hereinabove, the pRL-(UA6)₂ reporter with the UA6 Adaptor showedimproved knockdown (IC₅₀ of 2.2 nM) compared with the pRL-UA6 reporter(IC₅₀ of 7.4 nM), demonstrating the U1 Adaptor method shows additivesuppression if multiple binding sites exist on the same target. Incontrast, multiple siRNAs against the same mRNA do not result inadditive inhibition and instead show suppression at the level expectedfor the single most-potent siRNA in the pool (Hannon et al. (2004)Nature 431:371-8; Elbashir et al. (2001) Nature 411:494-8; Novina et al.(2004) Nature 430:161-4).

Optimization of U1 Adaptor Design

The UA6 Adaptor is a 25 nt LNA-DNA mixmer having 10 nt complementary tothe U1 snRNA and 15 nt complementary to the target. The hybridizationdomains in this U1 Adaptor are short yet function well due to the highLNA content of this oligonucleotide (15/25 bases are LNA). However,having a high LNA content increases the self-dimer and hairpin potentialof a sequence (which is further increased by the high stability ofLNA:LNA base pair events), complicating the design of U1 Adaptors whenapplied to other sites. Ways to decrease the relative LNA content wereexamined by comparing function of different chemistries and the lengthsof each domain using the UA6 Adaptor as a model system. First, an all2′OMe RNA version of the UA6 Adaptor was tested which showed reducedactivity (see hereinabove). However, the 10 nt U1 Domain could besubstituted with 2′OMe RNA with only a slight loss of activity (UA17-10,see hereinabove and FIG. 19). Continuing to use the 2′OMe RNA chemistry,a series of U1 Adaptors were synthesized varying the length of the U1Domain from 7 to 19 nt (see hereinabove and FIG. 19). As length of theU1 Domain decreased below 10 nt, activity was gradually lost. As lengthof the U1 Domain increased, activity increased and peaked at a length of13 nts. As length further increased, activity decreased. The UA17-13Adaptor having a 13 nt 2′OMe U1 Domain was 3-fold more potent than theoriginal UA6 Adaptor having a 10 nt DNA/LNA mixmer composition. Althoughit is not clear why U1 Domains longer than 13 nts show less activity, itmay be hypothesized that these longer sequences disrupt the foldingstructure of the U1 snRNA and may lead to decreased association with theU1-70K protein, the U1 snRNP subunit that inhibits polyA site activity(Gunderson et al. (1998) Mol. Cell 1:255-264). Similar results wereobserved with a U1 Adaptor specific for a different target sequence,demonstrating that a peak in activity for 13 nt U1 Domains is notpeculiar to UA6. In designing the UA17 series, it was assumed that theinhibitory activities of the UA17 Adaptors could be increased byincreasing their relative affinities to U1 snRNP. This was shown to bethe case by employing an EMSA competition assay (FIG. 20).

All of the U1 Adaptor sequences studied thus far had the Target Domainat the 5′-end and the U1 Domain at the 3′-end. Switching domain orderwas tested and it was found that U1 Adaptors having the U1 Domain at the5′-end were less effective than the original design (FIG. 21).Increasing the length of the U1 Domain to 13 nts did not improve potencyof the U1 Adaptor as much when using this configuration. A 2′OMe/LNAmixmer should have higher binding affinity than a uniform 2′OMe RNA orDNA/LNA mixmer sequence when hybridizing to an RNA target. Therefore,use of a mixed 2′OMe RNA and LNA sequence for the U1 Domain was tested,using the optimal 13 nt length. A variant of UA17-13 (the most potentAdaptor identified in FIG. 21 with an IC₅₀ of 1.5 nM) having five LNAnucleotides improved potency 3-fold and had an IC₅₀ of only 0.5 nM(UA17-13b, FIG. 22). These design improvements have therefore increasedpotency of the original UA6 Adaptor by over 10-fold.

Insights into design optimization of the U1 Domain described aboveshould apply to all U1 Adaptor oligonucleotides. Additional optimizationwas done examining similar design variation in the Target Domain.However, it is important to note that the optimal length, number andposition/configuration of modified nucleotides may vary for differenttarget sequences and so each new target gene and its target sequence mayrequire optimization of the U1 Adaptor's Target Domain. Versions of theUA6 Adaptor sequence having a 100%-2′OMe RNA Target Domain had reducedactivity (see hereinabove), whereas a 100%-LNA Target Domain slightlyincreased activity (see hereinabove). Sequences which are fully LNAmodified cannot activate RNase H, so these results rule out thepossibility that an RNase H antisense mechanism of action mightcontribute to the observed gene suppression. Assuming that higherbinding affinity is helpful, the length of the 100%-2′OMe RNA TargetDomain was increased incrementally from 15 nts to 25 and 35 nts and aloss of activity was observed. Although longer Target Domains might workat other sites, there does appear to be benefit from employing a short,high affinity sequence, which is most easily achieved using the LNAmodification. This may relate in part to target secondary structure, andsimilar findings have been reported for RNase H active antisenseoligonucleotides (ASOs); short, high affinity compounds are generallymore potent than long, low affinity compounds) (De Paula et al. (2007)RNA 13:431-56; Lennox et al. (2006) Oligonucleotides 16:26-42).Importantly, U1 Adaptors with a phosphorothioate (PS) backbone showedhigh potency (see hereinabove), a valuable feature as nucleaseresistance will likely be more important for function when U1 Adaptorsare tested in vivo. The ability of U1 Adaptors to inhibit target RNAswith less-than-perfect complementarity was assessed by testing variantsof UA17-13b having 1, 2 and 3 nt changes in the Target Domain against awt reporter and a mutated reporter having a compensatory 3 nt basechange in the target RNA. The results, shown in FIG. 23, demonstrate agraded response with a 3 nt mismatch having no activity and a 1 ntmismatch having around half the activity of the wt U1 Adaptor. Thus, interms of base mismatch discrimination, U1 Adaptors behave similarly tohigh affinity ASOs. Although ASOs can show single base discriminationwhen using low affinity (low Tm) modifications, like methylphosphonates,this level of specificity is usually not achieved when using highaffinity (high Tm) modifications like LNAs (Lennox et al. (2006)Oligonucleotides, 16:26-42; Giles et al. (1995) Nucleic Acids Res.23:954-61). Adaptors are unlike ASOs however, in that they should onlysuppress expression when tethering U1 snRNP to the 3′ terminal exon.Terminal exon restriction is a well-established property of U1snRNP-mediated inhibition of polyA sites (Beckley et al. (2001) Mol.Cell Biol. 21:2815-25; Fortes et al. (2003) Proc. Natl. Acad. Sci. USA,100:8264-8269; Liu et al. (2004) Nucleic Acids Res., 32:1512-7.). Asshown in FIG. 24, it was confirmed that U1 Adaptors have this sameterminal exon restriction by inserting U1 Adaptor binding sites in avariety of positions within a 3-exon/2-intron splicing reporter. Thus,any unintended cross-hybridization of U1 Adaptors to upstream exons andintrons is likely to have no effect on expression of that gene.

Inhibiting the Endogenous RAF1 Gene with U1 Adaptors

To assess the ability of U1 Adaptors to suppress expression ofendogenous genes, the UA25 Adaptor was designed to the human C-raf-1(RAF1) gene (NM_(—)002880). RAF I was selected because it is an oncogenewith potential therapeutic utility (Sridhar et al. (2005) Mol. CancerTher., 4:677-85). siRNAs are part of the RNA induced silencing complex(RISC) that includes RNA helicases thought to assist in silencing byunwinding target sequences that are hidden within secondary structures.In contrast, U1 snRNP intrinsically lacks RNA helicase activity andpresumably the Adaptor:U1 snRNP complex is unlikely to be capable ofrecruiting helicases, therefore target site accessibility will beimportant for optimal performance. Since ASOs have the same requirementfor target site accessibility, it seems likely that good antisense sitesmight also be good U1 Adaptor sites. The first RAF1 target site studiedwas therefore designed at a known potent antisense site in the terminalRAF1 exon previously identified (Monia et al. (1996) Nat. Med.,2:668-75). The UA25 Adaptor employs an 11 nt U1 Domain without LNAnucleotides (see hereinabove and FIG. 25) because longer U1

Domains (12 and 13 nts) with LNA residues resulted in a predicted highpotential for self dimer and hairpin structures at this precise site.

HeLa cells were transfected with the UA25 Adaptor and cell extracts wereanalyzed by Western blotting for RAF1 expression (FIG. 25). RAF1 proteinlevels were specifically reduced by UA25 Adaptor in a dose-dependentmanner. The control Adaptor UA25-mt, which has a 2 nucleotide mutationin the U1 Domain, was inactive. It has been reported that silencing ofRAF1 leads to cleavage of PolyA Ribopolymerase (PARP) as part ofinduction of apoptosis (Lau et al. (1998) Oncogene 16:1899-902).Re-probing the Western blot in FIG. 25 with an anti-PARP antibodydemonstrated that suppressing RAF1 using the UA25 Adaptor induces PARPcleavage (see hereinabove). Quantitative real-time PCR (qPCR)demonstrated that the observed reduction in RAF1 protein levelscorrelated with similar reductions at the mRNA level and based on thisan IC₅₀ of 8 nM was calculated (see hereinabove and FIG. 25). Incomparison, out of 34 ASOs analyzed in the Monia et al. study, the bestsequence, “ISIS5132”, had an IC₅₀ of 50 nM (Monia et al. (1996) Nat.Med., 2:668-75).

Three more anti-RAF1 U1 Adaptors were designed that targeted sites inthe terminal exon of RAF1 which were predicted to be uninvolved inunstructured areas of the mRNA and fit general antisense design criteria(McQuisten et al. (2007) BMC Bioinformatics, 8:184.). All threeinhibited RAF1 expression and were about 2-fold less active than UA25(FIG. 26). As functional data becomes available for a greater number ofU1 Adaptors, it may be possible to develop algorithms which predicteffective target sites. To support the generality of the U1 Adaptormethod, a second human gene, PCSK9, was targeted (FIG. 27). As shown,two anti-PCSK9 U1 Adaptors each silenced the target with an IC₅₀ in the4-5 nM range. Importantly, simultaneous targeting of PCSK9 with bothanti-PCSK9 U1 Adaptors gave enhanced inhibition, similar to what waspreviously observed for the Renilla reporter (see hereinabove). U1Adaptors by definition have two domains, however none of the experimentsso far have demonstrated the domains must be linked. To examine this,“half” Adaptors were tested that have either an isolated U1 Domain or anisolated Target Domain. As shown in FIG. 28, transfection of halfAdaptors either alone or together failed to inhibit the target genedemonstrating that the Target and U1 Domains must be linked forinhibition to occur. The requirement for an intact bifunctionaloligonucleotide to trigger suppression further argues againstinvolvement of any traditional antisense mechanism of action.

Combining U1 Adaptors with siRNAs Gives Enhanced Silencing

U1 Adaptors and siRNAs utilize distinct mechanisms of action that occurin different compartments of the cell (nucleus versus cytoplasm). Thustheir combined use to target a single gene would be predicted to giveadditive inhibition; additive inhibition has already been reported forthe combination of ASOs and siRNAs (Hemmings-Mieszczak et al. (2003)Nucleic Acids Res., 31:2117-26). To test this, the pRL-UA6 Renillareporter plasmid was targeted with an anti-Renilla siRNA (RL-siRNA) andthe UA17-13b Adaptor (FIG. 29). Co-transfection of RL-siRNA withUA17-13b improved inhibition as compared to use of the siRNA or U1Adaptor alone. Negative control oligonucleotides (Control siRNA and themutated UA7a Adaptor) did not reduce Luciferase expression. Thespecificity of this additive inhibition is shown by use of the pRLUA6revreporter that has the 15 nt UA6 binding site in reverse orientation. Asexpected, the RLsiRNA decreased expression of pRL-UA6rev, however theUA17-13b Adaptor had no effect on pRL-UA6rev expression either when usedalone or in combination with RL-siRNA. Lack of inhibition when thetarget site is in the inverted orientation, as with the UA6 Adaptor onpRLUA6rev, argues against repression being at the transcriptional levelor being mediated by the UA6 Adaptor binding to its target site in thedsDNA plasmid. Finally, analysis of additional U1 Adaptors unrelated toUA6 demonstrated that they also function synergistically with siRNA(FIG. 30).

To determine whether combining siRNAs and U1 Adaptors can similarlyenhance silencing of an endogenous gene, RAF1 was targeted bytransfecting UA25 and an anti-RAF1 Dicer-substrate siRNA (DsiRNA) (Roseet al. (2005) Nucleic Acids Res., 33:4140-4156; Kim et al. (2005) NatureBiotechnology, 23:222-226) either alone or together. Measurement of RAF1mRNA by qPCR demonstrated that the combined use of the U1 Adaptor andsiRNA resulted in enhanced silencing (FIG. 29). Western blots confirmedthat RAF1 protein levels were similarly reduced. Further, a similardegree of synergistic inhibition was observed when a siRNA and U1Adaptors were used to silence PCSK9 (FIG. 31). Thus, it can be concludedthat synergistic suppression is a general property when U1 Adaptors andsiRNAs are combined to target the same gene.

The potential for global off-target effects of the anti-PCSK9 U1Adaptors was assessed by microarray profiling, comparing themhead-to-head with an anti-PCSK9 siRNA. The results, shown in FIG. 32,indicate the two methods of gene knockdown have a very high degree ofoverlap (Pearson correlation of 0.93) suggesting the anti-PCSK9 U1Adaptors do not have any new off-target effect profile when comparedwith the anti-PCSK9 siRNA. The U1 snRNP complex is involved in splicingto produce mature mRNA. It is possible that binding of some U1 snRNPcomplexes with the U1 Adaptors might adversely affect splicing withinthe cell. The relative splicing patterns was examined of four endogenousgenes known to undergo alternative splicing and observed that theanti-PCSK9 U1 Adaptors had no discernable effect on the ratio ofalternatively spliced products for these four genes, at least withinHeLa cells (FIG. 33). U1 Adaptors are therefore unlikely to have aglobal effect on splicing, and this conclusion is further supported bythe data shown earlier using the splicing reporter constructs. Thedetermination of Adaptor specificity may be further studied withmultiple U1 Adaptors and multiple gene targets using global expressionprofiling techniques. The above improvements to U1 Adaptor potency anddesign parameters reduce the potential for off-target effects, somethingalready seen with siRNAs and ASOs.

U1 Adaptor Inhibition is at the mRNA Level

As a rigorous method to quantitate mRNA levels from Adaptor transfectedcells, a Ribonuclease Protection Assay (RPA) was used to measure RenillamRNA levels and normalized it to endogenous GAPDH mRNA levels. Asdescribed previously (Goraczniak et al. (2008) J. Biol. Chem.,283:2286-96), a Renilla RPA probe containing 100 nts of unrelated vectorsequence and 295 nts that span the Renilla coding region was used thatgave a 295 nt protected fragment (see FIG. 17). The Renilla-specificprotected probe was normalized to endogenous GAPDH mRNA detected using aprobe derived from a commercially available plasmid that gives a 307 ntprotected product (see FIG. 17). For each transfection as well as foruntransfected cells, the cells were split into two pellets, one tomeasure luciferase as was done in the main text and the other to maketotal RNA for RPA. The “5% Probe” lane is undigested probe at 5% theamount added to other lanes to show that the assay is in probe excess.To aid in quantitation and demonstrate that the assay is in the linearrange, the Renilla-specific RPA was repeated with varying amounts oftotal RNA as shown in FIG. 17. The RPA signals were quantified byphosphoimagery and normalized to the GAPDH RPA signal. The results asgiven in FIG. 17D indicate Renilla mRNA levels closely correlate withRenilla activity. Using a method to fractionate cytoplasmic and nuclearRNA (Goraczniak et al. (2008) J. Biol. Chem., 283:2286-96), cytoplasmicRenilla mRNA levels was found varied in the same way as in total RNApreparations. Thus the reduction in Renilla luciferase enzyme activityis primarily, if not completely, due to a reduction in mRNA levels.

U1 Adaptors Tether U1 snRNP to the Target mRNA

An Electrophoretic Mobility Shift Assay

(EMSA) was used to demonstrate that U1 Adaptors can tether U1 snRNPspecifically to a target RNA. As shown in FIG. 18, a ³²P-uniformlylabeled RNA (˜300 nt) called UA6-RNA derived from pRL-UA6 containing theUA6 binding site was mixed with highly purified HeLa cell U1 snRNP andeither the UA6 Adaptor or the UA7a negative control Adaptor and theresulting complexes resolved by native PAGE. The purification of HeLa U1snRNP and its use in EMSA is as described (Abad et al. (2008) NucleicAcids Res., 36:2338-52; Gunderson et al. (1998) Molecular Cell 1:255-264) wherein U1 snRNP specifically binds to RNA containing thesequence 5′-CAGGUAAGUA-3′ (a 10 nt U1 Domain; SEQ ID NO: 1) but not to amutated sequence 5′-CAacUcAcUA-3′ (mutations in lowercase; SEQ ID NO:76), the same mutation as found in UA7a (Abad et al. (2008) NucleicAcids Res., 36:2338-52; Gunderson et al. (1998) Molecular Cell 1:255-264). EMSA was done to confirm that U1 snRNP specifically binds aradiolabeled UA6 Adaptor but not UA7a. As shown in FIG. 18 lane 2, U1snRNP did not bind direct to the ³²P-UA6-RNA but was able to bind whenthe unlabeled UA6 Adaptor was present (lane 4). This binding depended onthe U1 Domain of UA6 as no complex was observed when the UA7a Adaptorhaving a mutated U1 Domain was used in place of UA6 (lane 6). Thus theUA6 Adaptor specifically tethers U1 snRNP to the UA6-RNA containing theUA6 binding site. Note that under these EMSA conditions, theAdaptor-³²P-UA6-RNA complex is not readily visible as it co-migrateswith the free 32P-UA6-RNA because the MW of Adaptors (<10 kDa) is farless than that of the 32P-UA6 RNA (˜100 kDa).U1 Adaptors May have Reduced Activity when the Target Domain is MadeEntirely of 2′-O-Methyl RNA (2′OMe)

It was analyzed whether U1 Adaptors with a 100%-2′OMe Target Domainmaintain potency. The UA6 and UA17-10 Adaptors were included as controlsand their comparison with UA-OMe1 indicates a 100%-2′OMe RNA U1 Domainmaintains potency whereas a 100%-2′OMe RNA Target Domain results inreduced activity. Lengthening UA-OMe1's Target Domain to 25 nts and 35nts failed to regain full activity. Thus, a Target Domain having eitheran LNA-DNA or LNA-RNA composition may be important for full U1 Adaptoractivity.

U1 Adaptor Activity Correlates with their Affinity to U1 snRNP

The UA17 series of Adaptors shown herein all have various inhibitoryactivities that are presumably due to different affinities to U1 snRNP.To directly test their relative U1 snRNP affinities, a competition assaybetween various unlabeled UA17 Adaptors and a ³²P-labelled RNA calledU1D-RNA having an 11 nt U1 Domain (U1D) was performed as shown in FIG.20. For all lanes, the amount of the U1 snRNP: 32P-labelled U1D-RNAcomplex was quantitated by phosphoimagery and is a measure of theability of the unlabeled UA17 Adaptor to compete for U1 snRNP bindingwith the absence of competitor (lane 2) being set to 100%. The UA17-7Adaptor could not compete for binding to U1 snRNP consistent with ithaving no inhibitory activity in cells. The increasing ability of theother UA17 Adaptors to compete, tightly correlates with their increasinginhibitory activity (see hereinabove). Thus the simplest explanation forthe various activities of the UA17 series is their different relativeaffinities to U1 snRNP.

U1 Adaptors with a 100% LNA Target Domain are Active

It was also determined whether U1 Adaptors with an all LNA Target Domainmaintain potency. To this end the UA24-12 and UA24-15 Adaptors thatmatch UA17-10 except they have a 12 nt or 15 nt all LNA Target Domain,respectively, were analyzed (see hereinabove). As shown these U1Adaptors were active on pRL-UA6 but not pRL-UA6rev indicating specificactivity is maintained when the Target Domain is comprised entirely ofLNA nucleotides. As complete conversion to LNA modified nucleotidesinhibit RNase H activity, it can be concluded that the mechanism ofthese Adaptors does not involve RNase H.

UI Adaptors with a Phosphorothioate (PS) Backbone are Active

Nucleotides with a PS backbone are commonly included in antisenseoligonucleotides (ASOs) as they increase half life in vivo by increasingnuclease resistance. The U1 Adaptors will likely have some improvednuclease stability compared with DNA due to incorporation of LNAresidues, however further stabilization of the U1 Adaptors with PSinternucleoside modifications would be predicted to improve function invivo, especially if intravenous administration is considered. Widespreadinclusion of PS bonds in siRNA decreases activity for this class ofgene-knockdown reagent; recent studies have shown, however, that limitedPS modification is compatible with active siRNAs (reviewed in Giles etal. (1995) Nucleic Acids Res., 23:954-61; Dahlgren et al. (2008) NucleicAcids Res., 36:e53). As the U1 Adaptor method does not requirecompatibility with any enzymatic activity, it was expected thatextensive PS modifications would not impair activity. To test thishypothesis, a set of U1 Adaptors that were fully PS-modified were testedas shown hereinabove. The results show that the PS backbone does notsignificantly impair U1 Adaptor activity.

Specificity Assessed by a Mutation/Compensatory Mutation Analysis

U1 Adaptor specificity is based on the Target Domain having perfectcomplementarity to a sequence within the target mRNA's terminal exon.However, it is unknown what level of inhibition would occur for mRNAswith varying degrees of less-than-perfect complementarity to the U1Adaptor. To assess specificity, three versions of UA17-13b were testedhaving 1, 2 and 3 nt changes in the Target Domain and observedincreasingly reduced activity (FIG. 23). A single base mutation causearound a 50% reduction in inhibition and the 3 base mutation wasinactive. A reporter called pRL-UA6-m3 was also made and tested that hasa 3 nt compensatory mutation that fully restores complementarity to theUA17-m3 Adaptor and found a full restoration of inhibitory activity.This cross validation analysis was continued by testing the activity ofUA17-ml and UA17-m2 on pRLUA6-m3 and it was found intermediate levels ofinhibition. In all cases a 3 nt mismatch abrogated inhibition, whereas a1 nt mismatch had partial to nearly full inhibition depending on whichU1 Adaptor was paired with which reporter, and 2 nt mismatches hadintermediate inhibition ranging between the inhibition seen for 1 and 3nt mismatches. Thus U1 Adaptors exhibit partial activity onless-than-perfect target sequences, a fact that is commonly observed forboth ASO-based silencing and for certain siRNAs. ASOs typically relyprimarily upon nucleic acid hybridization for specificity and there is atrade-off between “high affinity, high potency, lower specificity”reagents and “lower affinity, lower potency, higher specificity”reagents (Giles et al. (1995) Nucleic Acids Res., 23:954-61; Lennox etal. (2006) Oligonucleotides 16:26-42). Specificity for siRNAs isinfluenced by poorly understood interactions between the siRNA guidestrand, the mRNA target and Ago2 in RISC. Depending on the position ofthe base mismatch, siRNAs can show single base specificity or can showfull activity even in the face of several adjacent mutations (Dahlgrenet al. (2008) Nucleic Acids Res., 36:e53; Schwarz et al. (2006) PLoSGenet., 2:e140; Du et al. (2005) Nucleic Acids Res., 33:1671-7).

U1 Adaptors do not Effect Splicing of a Reporter Gene

As U1 Adaptors tether U1 snRNP, it is possible that unintendedcross-hybrdization to non-terminal exon regions within a pre-mRNA couldaffect splicing. As shown in FIG. 24, It was directly tested whether U1Adaptors can affect pre-mRNA splicing of a transcript that they aredesigned to basepair with. First, a splicing reporter plasmid (pFN) wasconstructed that contains a 3000 bp segment of the human Fibronectingene. The 15 nt UA6 binding site, that was sufficient to fully confer U1Adaptor inhibition to the pRLUA6 Renilla reporter, was inserted intofour distinct positions within pFN (FIG. 24A) including intronicpositions and the first and last exons. In all cases the reverseorientations were also included to test whether an affect would bemediated at the DNA level. A long intron was selected as this wouldallow for more time for annealing of the U1 Adaptor to the pre-mRNA soas to increase the likelihood of observing a U1 Adaptor mediated affect.The UA17-13b Adaptor was chosen as this is the most potent U1 Adaptor tothe UA6 binding site. Note that this segment of Fibronectin contains a273 nt alternatively spliced exon (Exon IIIB) that is included about 10%of the time in HeLa cells (see FIG. 33 for endogenous Fibronectin spliceisoforms). Exon IIIB inclusion could not be reliably detected whenperforming RT-PCR analysis of HeLa cells transfected with pFN, even when40 PCR cycles were used. In contrast, the major spliced product thatjoins Exon II17b direct to Exon III8a could be readily detected (FIG.24), thus it could be determined whether the UA17-13b Adaptor wouldaffect splicing. Each of the nine pFN-related plasmids were transfectedeither with the M13 control or with 5 nM of the UA17-13b Adaptor andafter 24 hours the cells were harvested and analyzed by RT-PCR. Giventhat the pRN-3for plasmid has the U1 Adaptor binding site in theterminal exon in the forward orientation, its splicing was expected tobe inhibited. As shown in FIG. 24B, lane 18 this was the case. Incontrast, none of the other pFN plasmids exhibited a change in theirsplicing pattern or efficiency when 5 nM UA17-13b was co-transfected.The primers were demonstrated to be specific for the pFN plasmid as noRT-PCR product was observed in the non-transfected cells (lanes 11 and22). For all samples, uniform RT-PCR bands were obtained for the Arf1housekeeping gene demonstrating the RNA samples and the RT-PCR were ofsimilar quality. Note that 5 nM UA17-13b Adaptor gives a 9.5-foldinhibition of the pRL-UA6 reporter that has the binding site in theterminal exon. Thus it can be concluded that any affect it may have isminor when not targeting the terminal exon. Finally, it should be notedthat these results are consistent with the prior work that mis-targetingU1 snRNP does not affect expression levels unless U1 snRNP is targetingthe 3′ terminal exon (Fortes et al. (2003) Proc. Natl. Acad. Sci. USA,100:8264-8269; Beckley et al. (2001) Mol. Cell Biol., 21:2815-25).

PARP Cleavage when RAF1 is Silenced

It is demonstrated herein that RAF1-specific silencing with theanti-RAF1 UA25 Adaptor. It has been shown that silencing of RAF1 leadsto induction of PolyA RiboPolymerase (PARP) cleavage as part ofinduction of an apoptotic pathway (Lau et al. (1998) Oncogene16:1899-902). To demonstrate that the anti-RAF1 UA25 Adaptor had thesame property, Western blotting was performed to visualize PARP. Theresults shown hereinabove demonstrate that the anti-RAF1 UA25 Adaptorinduces cleavage of PARP. Other Western blotting showed that PARPcleavage is dependent on the dosage of UA25 Adaptor used and closelyparallels the degree of silencing of RAF1 protein.

Additional Anti-RAF1 U1 Adaptors can Also Silence RAF1

To demonstrate silencing of RAF1 was not unique to the UA25 Adaptor,three more anti-RAF1 Adaptors called UA27, UA28 and UA29 were designedand tested as shown in FIG. 26. As was done previously, each Adaptor wastransfected into HeLa cells and cells were harvested after 24 hours andanalyzed by Western blot. The results demonstrate that the UA27, UA28and UA29 Adaptors can each silence RAF1 expression. Dose dependenceanalysis indicted these three Adaptors have an activity level about2-fold less than UA25.

“Half” Adaptors with Either Just the U1 or Target Domain are Inactive

Although it is likely that U1 Adaptors inhibit by tethering U1 snRNP togene-specific pre-mRNA, the data presented do not formally rule out thepossibility that the separate actions of each domain of the U1 Adaptorcauses inhibition of the target RNA. For example, the U1 Domain mighttitrate out some U1 snRNP and affect processing of the pre-mRNA,possibly “sensitizing” the mRNA to annealing of the Target Domain. Inaddition, the Target Domain “half” might trigger an antisense responseand lead to reduction of mRNA levels independent of the presence of U1snRNP. If this were the case, then unlinking the two domains to create“half” Adaptors should still result in gene specific inhibition. In FIG.28 the inhibitory activity of “half” Adaptors having either the TargetDomain or the U1 Domain were determined against the pRL-UA6 reporter(FIG. 28A) and against the PCSK9 endogenous gene (FIG. 28B). In neithercase were the half Adaptors capable of inhibition either whentransfected alone or together. Thus, U1 Adaptor activity requires thatthe Target and U1 Domains be covalently linked. To determine whetherinclusion of spacer sequences between the U1 and Target Domains wouldaffect function, a set of U1 Adaptors with spacer nucleotides ranging inlength from 2-6 nts was tested. In all cases such spacers did not have asignificant impact on activity. Given these results, it may be expectedthat non-nucleotide spacers will also support U1 Adaptor activity.

Enhanced Silencing of Renilla is Seen when Using a Different U1 Adaptorand an Anti-Renilla siRNA Together to Target a Renilla Reporter

Hereinabove, it was shown that the combined use of siRNA and theUA17-13b Adaptor gave enhanced silencing of a Renilla luciferasereporter. To demonstrate that this is not unique to the UA17-13b Adaptorand target site, these experiments were repeated in a different sequencecontext using the UA12 Adaptor that targets a site in the human GAPDH3′UTR. As shown in FIG. 30A, the UA12 Adaptor specifically inhibitsexpression of a Renilla reporter containing the GAPDH 3′UTR (calledpRLGAPDH) with an IC₅₀ of 1.8 nM. As shown in panel B, the combined useof an anti-Renilla siRNA and UA12 gave enhanced silencing of pRL-GAPDH,far better than when the siRNA and UA12 Adaptor were used alone.

Combining siRNAs and U1 Adaptors Gives Enhanced Silencing of anEndogenous PCSK9 Gene

To demonstrate enhanced silencing when the U1 Adaptor method is combinedwith siRNAs is also applicable to endogenous genes, human PCSK9 wastargeted using both U1 Adaptors and siRNAs. As shown in FIG. 31, thecombined use of anti-PCSK9 siRNA and an anti-PCSK9 U1 Adaptor resultedin enhanced levels of silencing when compared with silencing when eachmethod was used alone. Enhanced silencing is seen in other contexts whenboth methods are used together. Thus, the above supports the conclusionthat enhanced levels of silencing are seen when U1 Adaptors and siRNAsare employed together targeting the same gene.

Global Expression Analysis Comparing U1 Adaptors to siRNAs

The data presented so far show U1 Adaptors specifically silence a targetreporter plasmid as compared to a control reporter plasmid and silence atarget endogenous gene as compared to GAPDH. Such experiments do notaddress whether and to what degree U1 Adaptors affect the abundance ofnon-targeted mRNAs. Such off-target affects can arise from the U1Adaptor mistargeting either the polyA site regions of other genes orupstream exons or introns that would result in changes in the splicingpattern. As a first test to assess the global specificity of U1Adaptors, the anti-PCSK9 U1 Adaptors were compared head-to-head with theanti-PCSK9 siRNA by microarray profiling. The total RNA preparationsused in FIG. 31, namely the M13 control, the anti-PCSK9 DsiRNA and theanti-PCSK9 UA31d4+UA31e, were subjected to microarray analysis with theAffymetrix human U133 chip that detects ˜54,000 human mRNAs. A scrambledsiRNA control or a mutated control U1 Adaptor were not included as theoverall number of genes being effected by each method was assessedwithout normalization to a control. In order to do a global comparisonof the two methods, it was important that they have the same foldreduction in PCSK9. As shown in FIG. 32A, this was the case as qRT-PCRanalysis gave a 5.7-fold knock down for the anti-DsiRNA and a 5.1 foldknock down for the anti-PCSK9 U1 Adaptors. Importantly both themicroarray and qRT-PCR knockdown values were in good agreementvalidating the quality of the microarray data. If the anti-PCSK9 U1Adaptors and siRNA were perfectly specific, then in principle theirglobal expression profiles should be perfectly correlated. Panel B is acomparison plot of all the genes that showed ≧2-fold change for eitherthe U1 Adaptors or the DsiRNA. The line represents the ≧2-fold-affectedgenes from the U1 Adaptor transfection that are sorted from the largestincrease to the largest decrease. The vertical lines indicate thecorresponding genes from the DsiRNA transfection. If both the U1 Adaptorand siRNA methods were perfectly specific then one would expect thecurve and the vertical lines to perfectly overlap. A lack of correlationwould be, for example, when a U1 Adaptor gene is strongly up-regulatedwhile the DsiRNA gene is unaffected or downregulated. A visualinspection shows that there is a high degree of correlation between theU1 Adaptor- and DsiRNA-affected genes suggesting that the U1 Adaptormethod does not result in any larger degree of off-target effects thanresulting from using RNAi. Plotting the data as the log 2 ratio ofPCSK9-U1 Adaptor against the log 2 ratio of PCSK9-siRNA gives a Pearsoncorrelation of 0.93.

U1 Adaptors have No Apparent Effect on Alternative Splicing Pattern ofCertain Genes

Although microarray data gives a snapshot of the global mRNA expressionlevels it is less reliable at detecting changes in alternative splicingpatterns, especially if such changes affect minor spliced isoforms of agene. U1 Adaptors could affect splicing either by mis-annealing to anon-targeted pre-mRNA or by titrating out sufficient amounts of U1 snRNPso as to affect splicing in general. To address the latter four geneswere examined that are alternatively spliced in HeLa cells as suchsplicing involves suboptimal splice signals that should in principle bemore sensitive to reduced spliceosome activity caused by titrating outU1 snRNP by U1 Adaptors. It has recently been shown that the splicingpatterns of the alternatively spliced isoforms of the human Cdc25B andCdc25C genes are sensitive to changes in the levels of the canonicalU2AF35 splicing factor (Pacheco et al. (2006) Mol. Cell Biol.,21:8183-90). If the anti-PCSK9 U1 Adaptors were titrating out U1 snRNPthen this might lead to limitations in spliceosome complex formationthat would mimic a depletion in U2AF35 levels. As shown in the upper twopanels of FIG. 33, RT-PCR analysis of these genes showed no discernablechange in their splicing patterns. Two additional genes namely Grb2, asignal transduction gene, and Fibronectin, were analyzed where bothgenes have a minor isoform (Grb2's is exon 3-skipped and Fibronectin'sis Exon IIIB included) that increases in abundance during stress andthis increase is due to changes in splicing efficiency (Li et al., J.Biol. Chem., 275:30925-33; Kornblihtt et al. (1996) FASEB J.,10:248-57). As shown in FIG. 33, RT-PCR analysis demonstrated that theanti-PCSK9 U1 Adaptors had no discernable effect on the splicing patternof either Grb2 or Fibronectin as compared to either the M13 control orthe anti-PCSK9 siRNA. Thus it can be concluded that the anti-PCSK9 U1Adaptors are unlikely to have a global affect on splicing, a conclusionqualified by the fact that a concerted effort to measure all splicedisoforms in U1 Adaptor transfected HeLa cells could be done to morefully address this issue.

Comparison of Features Between U1 Adaptor, siRNA and ASO Methods

FIGS. 34 and 35 list the U1 Adaptor and siRNA sequences used in thisreport. FIG. 36 briefly summarizes what is known about the U1 Adaptormethod and compares it with siRNA and ASO gene silencing methods.

Discussion

A novel oligonucleotide-based gene silencing method called U1 AdaptorTechnology is described herein that reduces gene expression by tetheringthe U1 snRNP splicing factor to the target pre-mRNA. Successfulinhibition was demonstrated at both mRNA and protein levels and wasstudied for both a reporter gene and two endogenous human genes. Potentinhibition was observed with an IC₅₀ as low as 0.5 nM seen in the datapresented here. Potency in the subnanomolar range can routinely beachieved using this method. Within the limited set of U1 Adaptorsstudied so far, a success rate of approximately 50% was observed inobtaining U1 Adaptors with ≦5 nM IC₅₀ potency by applying antisenseoligonucleotide selection criteria to the target genes.

There are several considerations which support the prospect of using U1Adaptors in vivo and potentially for therapeutic indications. First, invivo administration of synthetic oligonucleotides such as U1 Adaptorscan employ the same delivery technologies already pioneered for use withsiRNA and antisense methods (Meister et al. (2004) Nature 431:343-9;Judge et al. (2006) Mol. Ther., 13:494-505; Soutschek et al. (2004)Nature 432:173-8; Morrissey et al. (2005) Nat. Biotechnol., 23:1002-7).Second, U1 Adaptors can include extensive modified nucleotides whichresult in molecules that are likely to have a high degree of nucleasestability, especially when phosphorothioate modified. Further, noenzymatic activity is involved in their function; this permits use of awider range of modifications in U1 Adaptors than are compatible withsiRNAs or traditional antisense oligonucleotides, which require directinteraction with cellular enzymes (Ago2, Dicer, RNase H, etc.) (Meisteret al. (2004) Nature 431:343-9; Manoharan, M. (2004) Curr. Opin. Chem.Biol., 8:570-9; Crooke, S. T. (2004) Curr. Mol. Med., 4:465-87). Third,the synergistic activity of several U1 Adaptors used together or incombination with siRNAs allows for use of lower doses of each individualoligonucleotide, reducing the potential for toxic side effects andlowering cost of administration. Importantly, the most active U1Adaptors described here were made entirely of 2′OMe RNA and LNAresidues; this chemical composition does not contain motifs that areknown to trigger the innate immune system. FIG. 36 summarizes acomparison of the U1 Adaptor method with antisense and RNAi methods.

Besides U1 snRNP, there are other RNA processing factors that are knownto inhibit polyA site activity and hence gene expression (Zhao et al.(1999) Microbiol. Mol. Biol. Rev., 63:405-45; Danckwardt et al. (2008)EMBO J., 27:482-98). Novel Adaptors could be designed to similarlyrecruit these other factors, either individually or in combination.However, there are several features unique to U1 snRNP. First, U1 snRNPis highly abundant with about 1 million copies present in a typicalmammalian nucleus (˜0.5 μM U1 snRNP in a HeLa cell, far higher in thenucleus) and is in ˜10-fold stoichiometric excess over the spliceosome(Will et al. (1997) Curr. Opin. Cell Biol., 9:320-8). Thus, withoutbeing bound by theory, it is plausible that sequestering a smallfraction of U1 snRNP by interaction with low nM amounts of U1 Adaptorswill have little effect on the overall splicing machinery and will notdeplete the pool of U1 snRNP available. Second, the functional in vivoconcentration of U1 snRNP, defined by the degree of inhibition observedwhen inserting a U1 snRNP binding site near a reporter gene's polyAsignal, is much higher when compared to these other RNA processingfactors (Fortes et al. (2003) Proc. Natl. Acad. Sci. USA, 100:8264-8269;Ko et al. (2002) J. Mol. Biol., 318:1189-206). Third, it is ratherstraight-forward to increase U1 snRNP's affinity with the U1 Adaptor asevidenced by the data herein. Nevertheless, new Adaptor designs can beidentified that inhibit gene expression by interaction with other RNAprocessing factors.

Example X

The ability to silence a single target gene while minimizing toxicityand off-target effects (OTEs) would offer the medical community a new,broadly-applicable tool to combat a wide variety of diseases bothprophylactically and therapeutically. A variety of hybridization-basedtechnologies have been tested that utilize synthetic nucleic acidoligonucleotides to suppress expression of a specific target gene. Thesetechnologies are, broadly speaking, based on a gene being targetedeither by a small interfering (si) RNA as part of the RNAi mechanism(Kim et al. (2007) Nat. Rev. Genet., 8:173-84; Castanotto et al. (2009)Nature 457:426-33; Krueger et al. (2007) Oligonucleotides 17:237-50) orby an antisense oligonucleotide (ASO) (Kurreck, J. (2003) Eur. J.Biochem., 270:1628-44) that can encompass a variety of mechanisms, butmost typically involve RNase H-mediated cleavage of RNA or sterichindrance of mRNA translation. ASOs and siRNAs have yet to produce acommercial therapeutic, in spite of initial hype and large-scaleinvestment (Castanotto et al. (2009) Nature 457:426-33). Therapeutic ASOhas struggled primarily because of modest potency and poor genome-widespecificity. Therapeutic siRNA although making tremendous progress sincesiRNAs were first described in 2001, suffers from immune activation (nowlargely solved), inherent poor stability and difficulty in delivery(still not solved), and general concern over adversely affecting thecell's natural microRNA pathway (not easy to solve).

The instant invention of U1 Adaptor gene silencing technologycircumvents these problems, as it represents a far more chemicallyflexible oligonucleotide platform, enabling generation of stable, potentmolecules with high specificity and ease of delivery. Unique features ofthe instant invention indicate that it will be far more potent thanother oligonucleotide-based silencing methods when used in vivoincluding therapeutic silencing. As their inhibitory mechanism does notrequire enzymatic activity, the U1 Adaptor oligonucleotides of theinstant invention can withstand up to 100% modification of theirbackbone and covalent attachment of delivery groups (e.g. peptides)enabling optimal stability and delivery capabilities. For example, thechemical composition of active U1 Adaptors can include 2′-O-Methyl(2′OMe) and phosphorothioate (PS) at every position as well 5′ or 3′ endmodification with bulky chemical groups such as biotin or fluorescentgroups (e.g., Cy3). This makes U1 Adaptors superior to siRNAs ortraditional ASOs that become inactive when so heavily modified (Kim etal. (2007) Nat. Rev. Genet., 8:173-84; Castanotto et al. (2009) Nature457:426-33; Kurreck, J. (2003) Eur. J. Biochem., 270:1628-44). Such achemically flexible platform will allow the U1 Adaptor to be more highlystable and deliverable in vivo compared to siRNA or ASO. An additionalfeature is the ability to observe additive and striking synergisticinhibition when multiple U1 Adaptors are used to simultaneously target asingle gene or when a U1 Adaptor and siRNA target a single gene,consistent with the fact that these two methods utilize distinctmechanisms of action (blocking pre-mRNA maturation versus destabilizingmature mRNA) that occur in different compartments of the cell (nucleusversus cytoplasm). This type of multiplexing to target a single genepermits deeper silencing while using less inhibitory molecules.

Here, the in vivo use of U1 Adaptor is demonstrated by targeting twohuman proteins that have been proposed to be important in melanoma in ahuman melanoma cell xenograft mouse model. The human B-cell lymphoma 2(BCL2) gene has long been a target for oligonucleotide-basedtherapeutics (Danial et al. (2004) Cell 116:205-219; Miller et al.(2006) N. Engl. J. Med., 355:51-65; McGill et al. (2002) Cell109:707-718; Zhao et al. (2007) J. Control Release 119:143-52; Loriot etal. (2010) Anticancer Res., 30:3869-78; Jansen et al. (2000) Lancet356:1728-33; Ocker et al. (2005) Gut 54:1298-1308; Okamoto et al. (2007)J. Cell. Mol. Med., 11:349-361; Yip et al. (2008) Oncogene 27:6398-6406;Gross et al. (1999) Genes Dev., 13:1899-1911; Jansen et al. (1998) Nat.Med., 4:232-234) whereas, the human metabolotropic Glutamate Receptor 1(Grm1) gene has more recently been established as a key player inmelanoma as well as other cancers including the finding that ectopicexpression of Grm1 in melanocytes is sufficient to induce melanocyticcell transformation in vitro and spontaneous melanoma development invivo (Pollock et al. (2003) Nat. Genet., 34:108-12; Shin et al. (2008)Pigment Cell Melanoma Res., 21:368-78; Lee et al. (2008) Pigment CellMelanoma Res., 21:415-428; Speyer et al. (2012) Breast Cancer Res.Treat., 132:565-573). A novel tumor-specific targeting vehicle suitablefor U1 Adaptor use in vivo is also provided. Unprecedented potency isdemonstrated herein with as little as 5.1 μg anti-BCL2 Adaptor needed toobserve 85% suppression of tumor volume in a xenograft mouse model ofmelanoma with little apparent organ toxicity. These results demonstratethat U1 Adaptor is a highly-potent gene-silencing therapeutic thatexhibits a tumor suppression potency far greater than otheroligonucleotide-based therapeutics.

Materials and Methods

Xenografts in Immunodeficient Nude Mice.

All animal studies were approved by the Institutional Review Board (IRB)for the Animal Care and Facilities Committee of Rutgers University. Malenude mice (5 weeks old) were purchased from Taconic (Hudson, N.Y.).Human melanoma cells, C8161 or UACC903, were injected into the dorsalarea at 10⁶ cells per site. Tumors were measured once a week with avernier caliper and tumor volume (V; cubed centimeters) was calculatedby using the equation V=d²×D/2, where d (squared centimeters) and D(centimeters) are the smallest and largest perpendicular diameters. Oncetumors reached 10 mm³, the animals were divided randomly into treatmentgroups so that the mean difference in tumor size between each group was<10%. Mice were sacrificed after approximately 3 weeks (for C8161) or 5weeks (for UACC903) corresponding to when the tumor volume in thevehicle-treated group had reached the maximum size permitted by the IRB.The tumor xenografts were excised for further histological and molecularanalyses.

Immunohistochemistry.

The Tissue Analytical Services at the Cancer Institute of New Jerseyperformed all the immunohistochemical staining of excised tumorxenografts to detect changes in apoptosis and proliferation usingwell-known activated Caspase 3 and Ki-67 markers, respectively.

Statistics.

The number of mice used for each experiment was determined with the helpfrom the Biometrics Facility Core at the Cancer Institute of New Jersey.P-values were determined using unpaired Student's two-tailed t-test. Ap-value of <0.05 was considered significant.

Results

The human BCL2 gene is a well-validated example of therapeutic targetingby ASO- and siRNA-based approaches (Waite et al. (2009) Bioconjug.Chem., 20:1908-16; Taratula et al. (2009) J. Control Release,140:284-93; Yip et al. (2008) Oncogene 27, 6398-6406; Namkoong et al.(2006) Front. Biosci., 11:2081-92; Okamoto et al. (2007) J. Cell Mol.Med., 11:349-61; Hwa et al. (2010) Pigment Cell Melanoma Res.,21:415-428; Gross et al. (1999) Genes Dev., 13:1899-911; Jansen et al.(1998) Nat. Med., 4:232-234). The human GRM1 gene in contrast has littleor no published examples of targeting by ASO and siRNA oligonucleotideswhile at the same time showing promise as a therapeutic silencing targetin that shRNA-based silencing of GRM1 leads to inhibition of tumorgrowth both in vitro and in vivo (Pollock et al. (2003) Nat. Genet.,34:108-12; Shin et al. (2008) Pigment Cell Melanoma Res., 21:368-78).Furthermore, both BCL2 and GRM1 have demonstrated potential for treatingmelanoma in that the extraordinary resistance of melanoma to apoptoticcell death commonly induced by anticancer drugs is mediated in part byelevated levels of BCL2 (Danial et al. (2004) Cell 116:205-219; Milleret al. (2006) N. Engl. J. Med., 355:51-65; McGill et al. (2002) Cell109:707-718), while GRM1's role in cancer was originally established inmelanoma and is now being therapeutically targeted by small moleculeinhibitors of GRM1. For xenograft mouse studies, the human C8161 cellline was used as a model of an aggressive melanoma.

Choice of an RGD-Dendrimer Delivery Agent

To deliver the U1 Adaptors to the tumor, the cyclic RGD pentapeptide(RGD) was used. The RGD peptide has a well-validated cancercell-specific targeting activity both in animal models and in humans.The RGD peptide targets a specific isoform (alpha-5 beta-3) of anintegrin cell surface receptor (arising from alternative splicing) thatis up-regulated in a wide variety of cancer types including melanomassuch as C8161 cells and is being used in several clinical trials as atumor-specific targeting moiety for MRI-visualization of tumors (PhaseI/II Study of SPC2996, an RNA Antagonist of Bcl-2, in Patients withAdvanced Chronic Lymphocytic Leukaemia (CLL); Wacheck et al. (2002)Antisense Nucleic Acid Drug Dev., 12:359-67). As a delivery backbone, apolypropyleneimine (PPI) generation 5 (G5) dendrimer was used for itssimplicity in the coupling of targeting moieties and that has beensuccessfully used in xenograft mouse models of human tumors (Cheng etal. (2008) Front. Biosci., 13:1447-71; Dufès et al. (2005) Adv. DrugDelivery. Rev., 57:2177-2202; Myc et al. (2010) Anticancer Drugs21:186-92). FIG. 37C is a schematic of the tumor-targeting deliveryvehicle wherein RGD was coupled to PPIG5 at a 2:1 stoichiometric ratioto make RGD-PPIG5 by use of a bi-functional SM(PEG)₁₂NHS linker (Pierce,Rockford, Ill.). For simplicity, unmodified PPIG5 is referred to as“Vehicle” and RGD-PPIG5 as “RGD-Vehicle”.

Anti-GRM1 Adaptors Silence GRM1 and Suppress Tumor Growth.

Screening a series of anti-human U1 Adaptors in C8161 cell cultureidentified three Adaptors, GRM1#1, GRM1#8 and GRM1#9 (FIG. 37A), ashaving superior potency to reduce GRM1 protein levels as compared to theproteins of two housekeeping genes, tubulin and GAPDH (used todemonstrate equal protein loading) as shown in the Western blot in FIG.37B. These transfections involved adding to the C8161 cells a complexcomprised of the U1 Adaptor combined with unmodified PPIG5 and then 60hours later lysing the cells in laemmli protein-gel loading buffer.Additional cultured cell studies established both strong nuclearlocalization of a Cy3-fluorescently labeled Adaptor and low cytoxicitywhen introducing into the recipient cells via transient transfection ofup to 100 nM Adaptor with either unmodified PPIG5 or RGD-PPIG5.

To assess their in vivo therapeutic activity each anti-GRM1 Adaptor wastested in a xenograft mouse system as follows. One million C8161 humanmelanoma cells were inoculated into both dorsal flanks of each nu/numouse. Mice were untreated until the tumor cell xenografts reached about10 mm³ (about 7-10 days), after which the mice were divided intotreatment groups 1-4 (TG1-TG4) such that each group had a similar meantumor volume. Each TG mouse received an intravenous (iv) injection ofeither the RGD-Vehicle only (the TG1 control group) or the RGD-Vehiclein complex with a specific anti-GRM1 Adaptor (TG2-TG4). Mice receivedtreatments twice weekly for 3 weeks until they were sacrificed when theTG1 tumor volumes reached the IACUC-allowed maximum size.

Tumor volumes were measured weekly and the results as graphed in FIG.37D demonstrate mice in the TG2, TG3 and TG4 treatment groups hadsignificantly smaller tumors as compared to the vehicle-only TG1 mice.To establish GRM1 is silenced in tumors treated with anti-GRM1 Adaptors,tumors were analyzed by Western blotting (FIG. 37D) and as the resultsdemonstrate, GRM1 protein levels were significantly reduced in TG2, TG3and TG4 mice relative to TG1 control mice using GAPDH and tubulin asnormalization for equal protein loading. Notably, having three validatedanti-GRM1 Adaptors allows for their combining to achieve additive orsynergistic tumor suppression. As discussed hereinabove, multiple U1Adaptors targeting a single gene result in deeper silencing activity(curiously, such activity is not observed with multiple siRNAs targetinga single gene). Having such potential in vivo will provide a means ofsignificantly lowering therapeutic doses.

Anti-BCL2 Adaptors Suppress Tumor Growth.

Having established the tumor suppression activities of the anti-GRM1Adaptors, an analysis of the human BCL2 gene was performed. BCL2 hasbeen implicated in a number of cancers, including melanoma, brain,breast, and lung carcinomas (Yip et al. (2008) Oncogene 27:6398-6406;Namkoong et al. (2006) Front Biosci., 11:2081-92; Okamoto et al. (2007)J. Cell Mol. Med., 11:349-61; Hwa et al. (2010) Pigment Cell MelanomaRes. 21:415-428; Gross et al. (1999) Genes Dev., 13:1899-911). Screeninganti-human-BCL2 Adaptors in cultured cells (including HeLa and C8161)identified the BCL2#11 and BCL2#12 Adaptors (see FIG. 38A) as havingsuperior potency (IC₅₀≦10 nM) to reduce human BCL2 mRNA as measured byqPCR normalized to two different housekeeping genes: HPRT1 and RPLPO.

The therapeutic activity of BCL2#11 in xenograft C8161 mice was assessedby i.v. injection that included varying the dosing amounts and times assummarized in FIG. 38B. TG5 was the control “vehicle-only” group, whereeach mouse was injected twice/week (Days 1, 5, 9, 12, 15) I.V. with 50μl of 1×PBS containing 2.3 μg RGD-Vehicle over a 2.5 week period givinga total of 5 injections. Each TG6 and TG7 mouse was injected I.V. on Day1 with 3.4 μg BCL2#11 Adaptor in complex with 4.6 μg RGD-Vehicle in 50μl 1×PBS. Then on Day 5, each TG6 and TG7 mouse was injected again with1.7 μg BCL2 #11 Adaptor in complex with 2.3 μg RGD-Vehicle in 50 μl1×PBS. TG6 mice received no further treatments. In contrast, each TG7mouse received three additional injections on Days 9, 12 and 15 day witheach injection comprised of 1.7 μg BCL2 #11 Adaptor in complex with 2.3μg RGD-Vehicle in 50 μl 1×PBS. All mice were sacrificed on Day 19 whenthe TG5 tumor volumes reached the IACUC-allowed maximum size.

Mice in both TG6 and TG7 had significantly smaller tumors as compared tothe vehicle-only TG5 mice with TG7 having higher efficacy in suppressingxenograft tumor progression. TG6 mice only received 2 injections with acumulative dose of 5.1 μg Adaptor/mouse (˜204 μg/kg), while TG7 micereceived 5 injections with a cumulative dose of 10.2 μg Adaptor/mouse(˜408 μs/kg). The potency of tumor suppression by the anti-BCL2#11Adaptor is impressive as the most potent anti-BCL2 siRNA in theliterature involved i.v. injection of 200 μg siRNA daily into each mouseover 24 days giving a cumulative dose of 4800 μg anti-BCL2 siRNA/mouse(Okamoto et al. (2007) J. Cell Mol. Med., 11:349-61). In spite of suchhigh amounts of anti-BCL2 siRNA, tumor suppression was a modest 30% ascompared to the 80-90% observed in FIG. 38B. The most potent anti-BCL2ASOs were even of lower potency involving cumulative dosing in xenograftmice in excess of 5 mg/mouse (Zhao et al. (2006) J. Control Release119:143-52; Loriot et al. (2010) Anticancer Res., 30:3869-78). Based onthese results, subsequent studies were performed with I.V. injection ofa given compound twice a week until the end of the experiments.

The experiments were repeated with additional controls to furtherestablish that tumor suppression was through a U1 Adaptor mechanism.FIG. 38C repeats the FIG. 38B experiment but simplifies the dosingregimem so all mice receive the same amount (1.7 μg) of anti-BCL2adaptor at the same frequency (2×/week). As can be seen, this simplerand lower amount of dosing was sufficient to give tumor suppression inTG9 as compared to the TG8 RGD-Vehicle only mice. As compared to othergene silencing methods, U1 Adaptors use a novel mechanism of action thatincludes a unique two-domain design in the oligonucleotide. Prior workin cell culture established design criteria for several control U1Adaptors that validate gene silencing is via a U1 Adaptor mechanismrather than via a traditional antisense mechanism. To this end, TG10animals were treated with a control BCL2#11 Adaptor containing a mutatedTarget Domain (still anneals to the U1 snRNP inhibitor but not to theBCL2 pre-mRNA), while TG11 animals used a control BCL2#11 Adaptor with amutated U1 Domain (still anneals to the BCL2 pre-mRNA but not to the U1snRNP inhibitor). In both TG10 and TG11, tumor suppression activity waslost, showing that tumor suppression is via a U1 Adaptor mechanism.

To demonstrate tumor suppression activity requires RGD targeting, TG12animals were treated the same as TG9 animals except the vehicle lackedthe RGD targeting group. As can clearly be seen, TG12 animals exhibitedno tumor suppression demonstrating tumor suppression depends on the RGDpeptide. TG13 mice were treated the same as TG9 except a 5-fold lowerdose of U1 Adaptor:RGD-Vehicle complex was used. The observation thatTG13 exhibited no tumor suppression indicates the minimal effective dosefor this xenograft system is ≦1.7 μg/dose and >0.34 μg/dose.

Therapeutic silencing of BCL2 by either antisense or siRNA establishedan increased rate of apoptosis as being part of the therapeuticmechanism (McGill et al. (2002) Cell 109:707-718 (2002); Zhao et al.(2006) J. Control Release 119:143-52; Loriot et al. (2010) AnticancerRes., 30:3869-78; Okamoto et al. (2007) J. Cell Mol. Med., 11:349-61) asassessed by immunohistochemical staining to visualize apoptotic markers.To establish whether the anti-BCL2 Adaptors elicited the same pattern,the tumors from TG8-TG11 mice (FIG. 38C) were analyzed by Ki67 andcaspase 3 staining with representative images shown in FIG. 39A andquantitation of the staining shown in the FIG. 39B graph. Comparison ofTG9 with the TG8 control mice identified elevated apoptotic activity(increased caspase 3 staining as seen in FIG. 39B) and reducedproliferation (lower Ki67 staining) consistent with the reduced tumorvolumes of the TG9 mice. The elevated apoptosis and lower proliferationindices were not found with the control Adaptor treated mice (TG10 andTG11) indicating they depend on simultaneous base pairing to BCL2 and U1snRNP. Thus U1 Adaptor targeting of BCL2 but not control U1 Adaptorselevates apoptosis, a result consistent with other silencingtechnologies that target BCL2.

A general concern in the oligonucleotide therapeutics field has beenwhether such treatments are toxic to organs or tissues. Indeed it hasbeen reported that use of milligram amounts of therapeuticoligonucleotides in mice can lead to organ toxicity. Given the far lowerU1 Adaptor dosaging levels used here, toxicity would likely not beapparent. Both visual and histopathological inspection of the livers andother organ systems (kidneys, spleen, heart, brain, lungs) from theTG8-TG13 mice indicated no overt toxicity. Furthermore, none of the miceexhibited overt signs of toxicity such as lethargy, not eating ordrinking, loss of body weight or ulceration of the transplanted tumor.

Targeting BCL2 with a Second U1 Adaptor.

To further confirm tumor suppression was through silencing of theintended BCL2 gene target and not some unrecognized off-target effect ofthe BCL2#11 oligonucleotide, a second anti-BCL2 Adaptor (BCL2#12) wastested that targets a different region of the BCL2 gene. As shown inFIG. 40, TG16 animals receiving BCL2#12 exhibited tumor suppression withnearly the same potency as BCL2#11 (TG15) consistent with theobservation in cultured cell studies where BCL2#11 is slightly morepotent than BCL2#12. Having such potency, when targeting a second sitein BCL2, indicates that tumor suppression is indeed acting through theBCL2 gene.

A simple method to increase oligonucleotide potency is to introducechemical modifications that increase stability and affinity to theintended target gene's pre-mRNA. Locked nucleic acid (LNA) is one suchmodification that increases affinity and to this end a series of secondgeneration variants of BCL2#11 containing LNAs in various positions werescreened in cultured cells and all demonstrated to have higher potencythan the original unmodified version of BCL2#11. The most effective ofthese (5-fold more potent than BCL2#11) was BCL2#11d2 that was thentested in TG17 mice (FIG. 40). Although BCL2#11d2 was active in tumorsuppression its efficacy was matched only to that of the originalBCL2#11.

To assess whether the linker itself was contributing to tumorsuppression we replaced the 53.4 angstrom (predicted length) linker witha different linker, LC-SPDP (Pierce) that is far shorter (predicted 15.6angstroms) and of a different chemical composition and tested for tumorsuppression activity in the TG18 mice. As can be seen the TG18 miceexhibited a similar tumor suppression as the matching TG15 mice whenboth are compared to the vehicle control TG14 mice indicating the typeof linker is not directly dictating tumor suppression activity.

To demonstrate tumor suppression is not limited to a single humanmelanoma cell line, that is C8161-derived tumors, a second humanmelanoma cell line was analyzed which harbors the most common B-RAFmutation (V600E) in melanoma, a mutation detected in about 70% ofsuperficial spreading and nodular cutaneous melanomas and is also oftenfound in benign, dysplastic nevi (Pollock et al. (2003) Nature Genetics,33:19-20; Davies et al. (2002) Nature 417:949-954). B-RAF^(V600E)containing melanomas are also refractory to therapy as the mutationconstitutively activates B-RAF's protein kinase activity producing asustained activation of the MAPK signaling cascade that in turn promotescellular proliferation and survival (Wellbrock et al. (2004) CancerRes., 64:2338-2342; Mercer et al. (2003) Biochim. Biophys. Acta.,1653:25-40). BCL2#11 suppressed UACC903 tumor xenografts growth in vivowith similar efficacy as detected for C8161 with wild type B-RAF (FIG.41). These results indicate that Adaptor mediated silencing of BCL2 hasbroad application to many types of melanoma independent of the genotypesat B-RAF.

To assess the effect of phosphorothioate (PS) modified nucleotides on U1Adaptors, TG21 mice used a PS-modified version of BCL2#11 (calledBLC2#11 PS3-3) that matches BCL2#11 but has 3 PS-modified bases at eachend of the oligonucleotide. As can be seen in FIG. 41, TG21 mice gavetumor suppression activity.

Example XI

Conjugates comprising a ligand (e.g., a cell-specific-targeting ligand)conjugated to a U1 Adaptor oligonucleotide of the instant invention areprovided. The ligand may be a synthetic molecule or derived from naturalsources. Examples of such ligands include, without limitation, a peptide(e.g. a linear peptide, a cyclic peptide), an antibody (e.g., monoclonalantibody of fragment thereof), a protein, a nucleic acid aptamer, apeptide aptamer, a spiegelmer, a lipid, carbohydrate, a carbohydratemimetic, a glycoprotein, small molecule, a metabolite or its derivativesand combinations of the above. The ligands may have at least twodesirable properties: 1) to have either no or at most a limited effecton U1 Adaptor activity as compared to a ligand-free Adaptor and, 2) toprovide cell type or tissue specific targeting (for example via receptorbinding) of the diseased cells while minimizing targeting of healthycells. Alternatively, the ligand may target to cell type or tissue inwhich the target gene is predominantly expressed in the body. Otherdesirable properties of a ligand are those shared with any therapeuticincluding for example to provide in vivo stability, clearance rate,bioavailability, and low or no toxicity. In some cases either a portionor the complete ligand may be covalently removed or cleaved from the U1Adaptor to further reduce the chances of effecting Adaptor activity.This could happen anytime after administration including after cellularuptake.

Some advantages of such conjugates include, without limitation: 1) Asimpler single-agent drug allowing for a simpler formulation that iseasier to manufacture at scale and to current Good ManufacturingProcesses (cGMP). 2) Simpler in vivo testing both in pre-clinical andclinical studies. For example, to establish an efficacious dosingregimen and toxicity with a two-component drug each component may haveto be tested alone in escalating doses and in combination with eachother and in various ratios one with the other. Multi-component drugsmay be more problematic in terms of pharmacokinetic/pharmacodynamic andtoxicity studies. 3) Permits lower doses of the Adaptor oligonucleotidedrug being administered.

Results

RDG-Adaptor conjugates were synthesized with the cyclic RGD (cRGD)pentapeptide as a tumor targeting ligand and an anti-BCL2 (BCL2-A;backbone: 5′-mGmCmCmGmUmAmCmAmGmUmUmCmCmAmCmAmAmAmGmGmGmCmCmAmGmGmUmAmAmGmUmAmU (m=2′O-methyl); synthesized by IDT Corp.(Coralville, Iowa)) or an anti-GRM1 (GRM1-A; backbone:5′-mA*mC*mG*mUmUmGmGmGmAmGmGmGmGmUmGmCmAmGmAmGmGmCmCmAmGmGmUmAmAmG*mU*mA*mU (m=2′O-methyl; *=phosphorothioate);synthesized by IDT Corp.) Adaptor as the active pharmaceutical genesilencing ingredient. FIG. 42 provides a general conjugation scheme. Thedescribed approach permits attachment of two cRGDs per U1 Adaptor or asingle cRGD and a free SH— group per U1 Adaptor as either ligandcombination is an effective tumor targeting approach. In other words,effective tumor targeting of the drug formulation would primarily bedirected by binding the alpha 5-beta 3 integrin receptor alone (Zitzmannet al. (2002) Cancer Res., 62: 5139-5143; Temming et al. (2006)Bioconjug. Chem., 17: 1385-1394; Haubner et al. (1997) Angen. Chem. In.Ed. Engl., 136, 1374-1389; Meyer et al. (2006) Curr. Pharm. Des.,12:2723-47; Ruoslahti, E. (1996) Annu. Rev. Cell Dev. Biol., 12:697-715;Reardon et al. (2011) Genes Cancer 2:1159-65; Desgrosellier et al.(2010) Nat. Rev. Cancer 10:9-22) or in conjunction with a free SH— group(Torres et al. (2012) Trends Biotechnol., 30:185-90; Hogg, P. J. (2003)Trends Biochem. Sci., 28:210-214; Tones et al. (2012) Nucleic AcidsRes., 40:2152-67). Because of these possibilities, conjugation ofmonomeric (cRGDm) and dimeric (cRGDd; catalog PCI-3651) cRGD forms withvarious conjugatable groups were tested, permitting one to controlstoichiometry in drug formulation.

cRGD binds its cognate receptor far more effectively as a dimer comparedto as a monomer, so pre-dimerized cRGD (i.e., cRGDd) may be more activein some applications than two cRGDm's tethered by a flexible linker.Monomeric (cRGDm; catalog PCI-3686; Peptides Intl. Inc. (Louisville,Ky.)) and dimeric (cRGDd; catalog PCI-3651; Peptides Intl. Inc.) cRGDforms with various conjugatable groups are commercially availablepermitting one to control stoichiometry in drug formulation. Both cRGDmand cRGDd were used herein.

cRGDm was conjugated to the 5′ end of atBCL2-A (amino terminal (NH₂) at5′ end of the BCL2-A Adaptor) through a two-step reaction using anLC-SPDP linker. Briefly, 2 nmoles atBCL2-A was added to freshly made 4nmoles LC-SPDP (Thermo Scientific) in 100 μl 20 mM Hepes pH 7.4 andreacted for 1 hour at room temperature. The pH was then lowered to 6.8and 6 nmoles (3 fold molar excess) of cRGDm was added in a volume of 200μl. The reaction proceeded for 1 hour and then an 8% PAGE analysis wasperformed. The final product is 10 μM RGD-Adaptor. Briefly, the 8M Urea8% PAGE analysis was performed using an 8% (24:1) denaturing 8M ureaPAGE that was run for 20 minutes (bromophenol blue marker migrated ˜10cm) and stained for 15 minutes with gel stain (Lonza (Allendale, N.J.))1:10,000. Samples were in 95% formamide with no dyes and heated for 2minutes at 95 degrees Celsius prior to loading.

Importantly, reaction efficiency could be readily monitored by analysison a denaturing 8M urea 8% PAGE that clearly separated products fromreactants and permitted a rapid and visual assessment of conjugationefficiency (FIG. 43). MALDI-TOF confirmed the molecular weights of thereaction products. As initial conjugations were noticeably inefficient(<10%), the linker:Adaptor and cRGD:SPDP-atBCL2-A ratios weresystematically increased, which led to increased conjugation efficiencyuntil it leveled off at about 30%. The gel photograph in FIG. 43 is anexample of a 30% efficient reaction. A similar 30% conjugationefficiency was observed when the atBLC2-A Adaptor was replaced with anatGRM1-1 Adaptor indicating this efficiency was independent of theTarget Domain sequence of the Adaptor.

As a means to improve reaction yield, thiol-based conjugation was used.FIG. 44 is a conjugation schematic using a dithiol (s₂) BCL2-A Adaptor,that was preferred over a monothio (s₁) version (single thio at 5′ endof the BCL2-A Adaptor), as that would permit conjugation of either twocRGD monomers or two cRGD dimers permitting a greater range of optionsfor in vivo experiments. Briefly, the conjugation protocol entailedpreparing reduced s₂BCL2-A such that it is converted to —SH form usingtris(2-carboxyethyl)phosphine hydrochloride (TCEP). 10 μl 0.5 mMs₂BCL2-A (5nmol, ˜60 μg) was added to 5 μl 1M Hepes 7.4 and Sμ10.2 MTCEP. The reaction was incubated for 30 minutes at room temperature. 80μl 50 mM Hepes 7.4 was added and the product was purified by gelfiltration using a ZEBA™ (Thermo Scientific) spin column. The productwas used immediately for conjugation below. For a 1:1.5 cRGDd:SMCCratio, 20 μl 1 mM (cRGD)₂ (20 nmoles) was added to 2 μl 1M Hepes 7.4; 3μl freshly made 5 mM LC-SMCC (Thermo Scientific; 30 nmoles); and 15 μlH₂O. The reaction was incubated at room temperature for 30 minutes. Fora 4× excess of SMCC-cRGD_(d) to reduced s₂BCL2-A, all of the TCEPreduced s₂BCL2-A was added to SMCC-cRGD_(d) and incubated at 4° C.overnight. 50 μl of 1×PBS added and un-reacted SMCC-cRGDd was removed bygel filtration on 0.5 ml Zeba™ spin columns. A PAGE analysis was thenperformed as described hereinabove.

FIGS. 45 and 46 provide examples where the ratios of reactants werevaried to increase efficiency. Further analysis established that up to60-70% conjugation efficiency was routinely obtained with two examplesprovided in FIG. 46A, lane 4 and FIG. 46B, lane 2. Additionaloptimization may be contemplated to increase reaction yields stillfurther.

In FIG. 47, a PEGylated variant of the cRGDm monomer called cRGD-PEG(cyclic RGD monomer-(PEG)₂ (Peptides Intl. Inc., Louisville, Ky.,catalog PCI-3696)) was conjugated to the adaptor as PEG is often used toimprove drug potency in vivo. FIG. 47 is an example of a 1.4 mg scalecRGD-PEG conjugation where the efficiency was around 30%. Briefly, 250μl 0.5 mM s₂BCL2-A (0.125 moles); 40 μl 1M Hepes 7.4; 25 μl 0.2 M TCEP(2.5 moles); and 75 μl H₂O were incubated for 3 hours at 4° C. 1000 μl 5mM cRGD-PEG (5 moles); 27.5 μl 1M Hepes 7.4; 2.8 μl EDTA; and 375 μlfresh 20 mM LC-SMCC (7.5 moles) were incubated at room temperature for 1hour. 390 μl (0.125 moles) of reduced s₂BCL2-A was then added andincubated at 4° C. overnight. Un-reacted cRGD-PEG-LC-SMCC was removed bygel filtration on 5 ml Zeba™ spin column and the sample was dialyzedinto 1×PBS.

To assess whether conjugation had a deleterious effect on Adaptorsilencing activity, the cRGD-PEG-s₂BCL2 (FIG. 47) conjugate in complexwith Lipofectamine™ 2000 (LF2000) was transfected into C8161 cells.Given the conjugate contained about 30% unconjugated Adaptor, BCL2-A incomplex with LF2000 was also transfected as a control. The results asshown in FIG. 48, indicate the conjugate and BCL2-A had the samesilencing efficiency, indicating the attachment of cRGD-PEG had nodetectable detrimental effect on silencing in vitro (see abovedescription of methods). It was then determined whether thecRGD-PEG-s₂BCL2 Adaptor, via its RGD ligand, would be able to enterC8161 cells and silence BCL2 mRNA without LF2000. Indeed, this was thecase as 100 nM and 300 nM cRGD-PEG-s₂BCL2 transfected alone with novehicle silenced BCL2 mRNA, a potency consistent with the preparationcontaining only ˜30% of the full cRGD-PEG-s₂BCL2 species. Transfectionof 100 nM and 300 nM BCL2-A alone gave no silencing of BCL2. Theseresults indicate the cRGD-PEG group has conferred a new property, namelythe ability to deliver the silencing activity of BCL2-A into cells inthe absence of a delivery vehicle.

The cRGD-PEG-s₂BCL2-A preparation from FIG. 47 was next tested for itsefficacy in suppressing human tumor growth in C8161 xenograft mice usingthe same treatment protocols as described hereinabove. The results, asshown in FIG. 49, demonstrate cRGD-PEG-s₂BCL2-A has an impressivelow-dose tumor suppression activity. This activity is not due tounconjugated s₂BCL2-A as non-targeted BCL2-A Adaptor administered tomice at single digit microgram doses lacks tumor suppression activity.Therefore, the tumor suppression activity is due to the intermediate(cRGD-PEG)₁-s₂BCL2-A or complete (cRGD-PEG)₂-s₂BCL2-A products of theconjugation reaction.

Conditions were also identified for the purification of thecRGD-PEG-s₂BCL2-A preparations that cleanly separated the variousconjugation products at both an analytical (FIG. 50) and a preparative(FIG. 51) scale. For FIG. 51, Peaks A-E yielded 208 μg, 84 μg, 146 μg,278 μg, and 55 μg. Briefly, samples were purified over a Hypersil™ ODS(C18) column (Thermo Scientific) with Solution A: 0.1 M triethylammoniumacetate (TEAA), pH 7.0; Solution B: 100% acetonitrile; and a gradient10-30%, 1 ml/min. MALDI-TOF mass spectrometry confirmed the molecularweights of the reaction products. The peaks seen in FIGS. 50 and 51(e.g., Peaks C and D) may be tested in C8161 xenograft mice to determinetheir tumor suppression activity. Such experiments may be performed withadditional controls as described hereinabove including conjugatescontaining: 1) an inactive Adaptor having a mutated Target Domain, 2) aninactive Adaptor having a mutated U1 Domain, and 3) an inactive versionof the cRGD peptide (e.g., RAD peptide).

Additional conjugation chemistries may also be used to create theconjugates of the instant invention including for example “CLICKchemistry” involving a copper-based catalysis between a U1 Adaptorcontaining one or more alkyne groups (for example hexanyl) and theligand containing one or more azides. Copper-free CLICK conjugation mayalso be performed using, for example, DBCO derivatives. CLICK chemistryshould, in principle, provide for faster reaction kinetics and a farhigher conjugation efficiency thereby obviating the need for extensivepost-conjugation purification steps.

In addition to the above, antibodies which target tumors, in particulartherapeutic monoclonal antibodies, may be conjugated to the Adaptors.Herceptin® (trastuzumab), a monoclonal antibody, was chosen as anexample as it has long been used in the clinic to treat certain forms ofbreast cancer. Indeed, recent clinical trials of breast cancer patientsinvolving a drug where chemotherapeutic agents are conjugated totrastuzumab have shown such conjugates provide a therapeutic benefitbeyond use of just trastuzumab alone (Junttila et al. (2011) BreastCancer Res. Treat., 128:347-56). Accordingly, the tumor suppressionactivity of trastuzumab can be enhanced by having it deliver U1 Adaptorsinto cells. Upon entry, the trastuzumab-Adaptor conjugate will undergoantibody processing resulting in release of the U1 Adaptor that can thensilence its target gene.

Briefly, the protocol for conjugating the antibody to the adaptorfollows what was done in FIG. 47 for the cRGD-PEG-s₂BCL2-A prep, withthe following changes. Trastuzumab was reacted with 3 molar excess ofSMCC followed by reaction with varying amounts of reduced s₁BCL2-A(single thio) at a 1.5, 4 and 12 molar excess to trastuzumab. Theproducts were then dialyzed in 1×PBS and protein concentration (BioRad)and conjugation efficiency was assessed by SDS-PAGE. The final yieldsfor TB (trastuzumab:s1BCL2-A) conjugates were HB 1:1.5-4.2 mg/mltrastuzumab, 0.5 mg/ml s₁BCL2-A in 1×PBS; TB 1:4-1.4 mg/ml trastuzumab,0.5 mg/ml s₁BCL2-A in 1×PBS; and TB 1:12-0.5 mg/ml trastuzumab, 0.5mg/ml s₁BCL2-A in 1×PBS.

SDS PAGE analysis of the preparation demonstrated that conjugation hadoccurred (FIG. 52). Notably, the 1:4 ratio may be desirable forconjugation as it may be undesirable to have too many Adaptors pertrastuzumab. The tumor suppression activity of these trastuzumab-Adaptorconjugates may be tested in C8161 xenograft mice.

In addition to the above, human serum albumin (HSA), a widely usednanocarrier (Lochmann et al. (2005) Eur. J. Pharm. Biopharm.,59:419-429; Abbasi et al. (2011) Cell Biochem. Biophys., 61:277-87;Kratz, F. (2008) J. Controlled Release, 132:171-183), was conjugated toBCL2-A and cRGD. Briefly, ˜25 LC-SPDP linkers were attached to freeamino groups found on lysines of HSA. Subsequently, ˜7 copies of singlethio-(s₁)-BCL2-A Adaptor were conjugated to the linkers followed byconjugation of ˜7 copies of the cRGDm to the remaining linkers. Anyunreacted linkers were inactivated by blocking with cysteine. Morespecifically, the single surface thiol group on HSA was blocked asfollows: 1.2 ml (60 mg, 0.9 μmole) HSA in H₂O, 18 μl 500 mM L-Cysteinein H₂O (9 μmoles), 60 μl 1 M Na-phosphate pH 7.2, was incubated at RTfor 2 hours and then dialyzed against 2×1L H₂O to remove cysteine.Measure A₂₇₉: 1 OD of HSA=1.883 mg. 6 μl HSA-Cys (6.6.12)+294 μl water:OD₂₇₉=0.288×1.883×50=27.1 mg/ml (51.5 mg in 1.9 ml) 0.4 mM HSA-Cys inH₂O (6.6.12). Stored at −20° C. HSA was activated with LC-SPDP asfollows: 100 μl 0.4 mM HSA-Cys (6.6.12) (40 nmoles, 2.7 mg), 860 μl 0.1M Na-phosphate buffer (pH 7.2), 40 μl 50 mM LC-SPDP (2000 nmoles) wasincubated for 3 hours at RT and then dialyzed 2× with water at RT (2 heach) and 1× with PBS overnight at 4° C. Measure volume after dialysis:1.8 ml, 22 μM HSA-SPDP (1.5 mg/ml). A pyridine-2-thione assay wasperformed to determine the HSA-SPDP ratio. Briefly, 100 μl 22 μMHSA-SPDP was added to 900 μl H₂O plus 10 μl DTT 15 mg/ml. The mixturewas incubated 15 minutes. OD₃₄₃=0.45 nmoles of release P2T in wholeprep: 0.45/8.08×1000×18=1002 nmoles. Ratio of HSA to SPDP was 1:25.

s₁BCL2-A was reduced to —SH form as follows: 1 ml of immobilized TCEP(Thermo Scientific #77712) was washed 4 times with 1 ml TNE (20 mMTrisHCl pH7.5, 100 mM NaCl, 1 mM EDTA). TCEP-gel was resuspended in 1 mlTNE and 480 μl 0.25 mM s1BCL2-A (0.120 μmoles, 1.37 mg) was added. Themixture was incubated 1 h at RT on rotary wheel. The mixture was spunfor 1 min at 1000 g and the supernatant was collected. Again, the gelwas resuspended with 500 μl TNE, the supernatant was collected and thencombined with the first one. OD at 260 nm was measured and yield wascalculated to be 108 nmoles (1232 μg) of reduced s₁BCL2-A in 1.85 mlTNE. The conjugation of s₁BCL2-A to HSA-SPDP was performed as follows:0.675 ml 19.4 μM HSA-SPDP (15 nmoles) was added 0.325 ml TNE.OD₃₄₃=0.021. 1.85 ml reduced s₁BCL2-A (108 nmoles) was added. OD at 343nm: after 1 min=0.065, after 10 min=0.1, after 1 h=0.24 and after o/nincubation at +4° C.=0.325. nmoles of attached s₁BCL2-A was calculated:(0.32−0.02)/8.08×1000×2.85=106 nmoles). HSA to s₁BCL2-A ratio was 1:7.The conjugation of cRGD to HSA-s₁BCL2-A-SPDP was performed as follows:50 μl 1 M Tris HCl pH 8.0 was added to 12 μl 10 mM cRGD (120 nmoles) in3% acetic acid. OD at 343 nm after 3 h incubation at RT was 0.63.Nanomoles of attached cRGD were calculated:(0.63−0.32)/8.08×1000×2.9=111 nmoles. HSA-s₁BCL2-A to cRGD ratio was1:7.4. Unreacted LC-SPDP was blocked as follows: 2 μl 500 mM Cysteinewas added to block remaining active SPDP. After 1 hour incubation at RT,OD₃₄₂=1.1. Total nmoles of P2T was calculated as 1.1/8.08×1000×2.9=395.Max should be 375 nmoles. Accordingly, all SPDP was saturated. Themixture was dialyzed 2× with 1×PBS overnight at 4° C. The prep wasHSA-(cRGD)₇-(s₁BCL2-A)₇ with HSA:cRGD:s₁BCL2-A ratio=1:7:7.4. Forsimplicity the ratio is rounded to 7. The volume was 2.8 ml, HSA was 5.3μM (0.36 μg/μl) and the s₁BCL2-Adaptor is 38 μM (0.44 μg/μl).

Note the SPDP linker has a reversible S—S bond that will release theAdaptor payload upon entry into cells. Some of the more notableadvantages of this system are the increased payload of Adaptors andcRGDs per particle and that HSA as a nanocarrier is expected to havevery little toxicity in vivo. While cRGD monomers are shown, cRGD dimersmay be used. Further, free SH— groups may be attached as describedhereinabove. Fluorescent groups may also be added to track tumorlocalization in vivo.

In addition to the above, chimeric oligonucleotides comprised of nucleicacid aptamers covalently linked to gene-specific U1 Adaptors can besynthesized. Nucleic acid aptamers can be naturally occurring. However,most nucleic acid aptamers are derived from diverse combinatoriallibraries using Systematic Evolution of Ligands by Exponentialenrichment (SELEX) as well as a cell-based version called Cell SELEX(Sundaram et al. (2013) Eur. J. Pharm. Sci., 48:259-71; Burnett et al.(2012) Chem Biol., 19:60-71; Magalhães et al. (2012) Mol. Ther.,20:616-24; Shigdar et al. (2011) Cancer Sci., 102:991-8; Thiel et al.(2012) Nucleic Acids Res., 40:6319-37). Typically, such aptamers exhibitcell type selectivity most typically by recognition of cell surfaceproteins (receptors) as well as other cell surface features. Theattractiveness of such aptamers is the simplicity of appending them totherapeutic oligonucleotides such as siRNAs and ASOs. A significantdisadvantage, however, is aptamers typically inactivate the genesilencing activity of the siRNA or ASO to which they are attached,presumably due to interference with RISC and RNase H, respectively. Incontrast, U1 Adaptors are unlikely to be inactivated when attached toaptamers.

As a test case, the cell penetrating aptamer called “C1” as described in(Magalhães et al. (2012) Mol. Ther., 20:616-24) was appended to theBCL2-A U1 Adaptor to make C1/BCL2-A. As shown in FIG. 53, transfectionof C1/BCL2-A in A549 cells with lipofectamine demonstrated the chimerahad silencing activity similar to the parent BCL2-A U1 Adaptor.Importantly, transfection of the chimera with no vehicle gave similarsilencing activity indicating the C1 domain was functional to deliverthe BCL2-A U1 Adaptor into cells. As expected, transfection of theBCL2-A with no vehicle gave no silencing activity. The overall resultindicates the chimera is effective.

Lastly, U1 Adaptors may be synthesized with small chemical groups thatfacilitate cell-specific targeting. For example, a biotin group may beattached as part of targeting to therapeutic antibody complexes,including for example radioimmune therapy formulations that would homethe U1 Adaptor to the tumor type. The presence of non-polar fluorescentgroups can also facilitate U1 Adaptor uptake into cells. An example ofthis is shown in FIG. 54, where a Cy3-labeled non-specific U1 Adaptor(Cy3-NC3wt) when directly delivered into mouse brain viaintraparenchymal injection resulted in rapid diffusion (two hourspost-injection) from the injection site and penetration into mouse brainneurons leading to a striking localization within spiny neurons as wellas other neighboring cells (FIG. 54). More specifically, 80 ng ofCy3-NC3wt U1 Adaptor in 1.5 μl H₂0 over a 10 minute interval wasintracerebrally injected into a female mouse (C57BL6) brain striatumusing a stereotaxic injection device. After two hours the mouse wassacrificed and the brain perfused with paraformaldehyde followed by thinsectioning and confocal analysis. The Cy3-labeled U1 Adaptor is stronglylocalized in nuclei of spiny neurons as well as other cells near theinjection site. Cy3-Adaptors diffuse out from injection site and giveevidence of a gradient of cytoplasmic localization for cells far frominjection site, and nuclear localization for cells near the injectionsite. While this type of nuclear accumulation with a Cy3-labeled U1Adaptor is also observed in vitro, the kinetics are slower andfluorescent signal intensity lower when compared to what is shown invivo in FIG. 54.

Example XII

Anti-KRAS U1 adaptor-iRGD peptide conjugates and anti-KRAS U1adaptor-cRGD peptide conjugates were designed and tested againstpancreatic tumors in vivo. The human KRAS gene is expressed in two mRNAforms: a major variant (GenBank Accession No. NM_(—)004985; 5312 nts)and a minor variant (Exon 4a) that code for different KRAS proteinisoforms. Both variants have the same terminal exon. Accordingly, U1Adaptors targeting the terminal exon will silence both variants.

Eight anti-KRAS U1 Adaptors were synthesized that target variouspositions in the terminal exon. These anti-KRAS U1 Adaptors are:

KRAS-1:mAmUmAmGmAmAmGmGmCmAmUmUmAmUmCmAmAmCmAmC-mGmCmCmAmGmGmUmAmAmGmUmAmU;KRAS-2:mAmGmUmCmUmGmCmAmUmGmGmAmGmCmAmGmGmAmAmA-mGmCmCmAmGmGmUmAmAmGmUmAmU;KRAS-3:mUmGmCmAmCmCmAmAmAmAmAmCmCmCmCmAmAmGmAmC-mGmCmCmAmGmGmUmAmAmGmUmAmU;KRAS-4:mAmAmUmAmGmCmAmGmUmGmGmAmAmAmGmGmAmGmAmC-mGmCmCmAmGmGmUmAmAmGmUmAmU;KRAS-5:mUmUmUmGmGmGmGmAmGmAmGmUmGmAmCmCmAmUmGmA-mGmCmCmAmGmGmUmAmAmGmUmAmU;KRAS-6:mUmCmUmGmAmCmAmCmAmGmGmGmAmGmAmCmUmAmCmA-mGmCmCmAmGmGmUmAmAmGmUmAmU;KRAS-7:mUmAmGmUmCmCmCmUmCmCmCmCmAmUmUmUmUmGmAmC-mGmCmCmAmGmGmUmAmAmGmUmAmU; andKRAS-8:mCmAmCmCmAmCmCmCmCmAmAmAmAmUmCmUmCmAmAmC-mGmCmCmAmGmGmUmAmAmGmUmAmU;where m=2′-O-methyl and the underlined is the U1 domain.

The panel of anti-KRAS U1 adaptors were transfected into humanpancreatic carcinoma MIA-PaCa2 cells using 150 nM PAMAM-G5 dendrimer asthe vehicle. The U1 Adaptor and dendrimer complex was formed asdescribed above (see also Goraczniak et al. (2013) Mol. Ther. NucleicAcids 2:e92). After 72 hours, a qPCR analysis was performed to measurethe amount of KRAS mRNA in the cells. As seen in FIG. 55, KRAS-2 andKRAS-3 had superior silencing activity. Western blotting analysisconfirmed the reduction in KRAS protein.

As shown hereinabove, cyclic-RGD-dendrimers can deliver anti-BCL2 U1Adaptors resulting in suppression of tumor growth in xenograft mice.Similar studies were performed with the two anti-KRAS U1 Adaptors(KRAS-2 and KRAS-3). Specifically, a MIA-PaCa2 subcutaneous xenograftmouse model was treated twice per week for 4 weeks by intravenousadministration of 0.16 mg U1 Adaptor/kg. As seen in FIG. 56, xenografttumor volume was reduced when KRAS-2, KRAS-3, or BCL2-A (positivecontrol) were administered in complex with cRGD-dendrimer vehicle.

To avoid the use of dendrimers, which can be challenging to manufactureaccording to cGMP requirements, tumor targeting peptides were conjugateddirectly (or via a linker) to U1 Adaptors. Click chemistry was used toconjugate a tumor targeting peptide to the U1 Adaptor. In a particularembodiment, the click chemistry is the Huisgen 1,3-dipolar cycloadditionlinkage of azides and terminal alkynes (Kolb et al. (2003) DrugDiscovery Today 8:1128-1137; Hein et al. (2008) Pharm. Res., 25:2216-2230). Here, a click chemistry using a copper-based catalysisbetween an alkyne groups (for example hexanyl) and an azide wasperformed. For example, the peptide can be synthesized with an azidogroup (such as for iRGD; the azido group may be attached via a linker(e.g., PEG)). Then a one step conjugation process can be used byperforming click coupling with an alkyne (e.g., hexanyl) containing U1Adaptor. If the peptide lacks an azide group after synthesis, then theazide group can be added via a conjugatable group such as a conjugatablethio group. For example, a thio group of the peptide can be reacted withNHS-linker (e.g., PEG₄)-azido to generate an azide containing peptide.The azide labeled peptide can then undergo a click coupling reactionwith an alkyne labeled U1 Adaptor. Notably, the alkyne group can beadded to the 5′ end and/or 3′ end of the U1 Adaptor to allow for theattachment of the peptide at the 5′ end and/or 3′ end, particularly atthe 5′ end.

Here, to conjugate cRGD₂ (catalog PCI-3651; Peptides International,Louisville, Ky.) and azido-PEG₄-NHS, 75 μl 10 mM cRGD2, 20 μl 1Mphosphate buffer pH 7.2, and 30 μl 100 mM azido-PEG₄-NHS (AZ103, ClickChemistry Tools; Scottsdale, Ariz.) were incubated overnight at 4° C. Asample was run on a C18 HPLC column (4.6×250 mm) with solvent A: 0.1%TFA and solvent B: 0.1% TFA, 99.9% acetonitrile with a gradient of 5-45%over 40 minutes at 1 ml/min. Purified (cRGD)₂-PEG₄-azide was isolatedfrom fractions corresponding to the product-containing peak and dried ina speed vac for storage as a powder.

Click conjugation was used to produce U1 Adaptor-peptide conjugates withthe BCL2-A U1 Adaptor, KRAS-2 U1 Adaptor, or KRAS-3 U1 Adaptor and thetargeting ligand cRGD monomer or cRGD dimer. The U1 Adaptor-cRGD peptideconjugates were shown to be very pure by stained 8% denaturing PAGE orHPLC chromatogram.

More specifically, a copper(I)-catalyzed azide-alkyne cycloaddition(CuAAC) reaction based on Hüisgen 1, 3 dipolar cycloaddition was used.Typically, the reactions require simple or no workup or purification ofthe product. The most important characteristic of the CuAAC reaction isits unique bioorthogonality, as neither azide nor terminal alkynefunctional groups are generally present in natural systems. The use ofthis method for DNA/RNA modification has been somewhat delayed by thefact that copper ions damage DNA/RNA, typically yielding strand breaks.These problems can be overcome by the use of copper(I)-stabilizingligands (e.g., tris(benzyltriazolylmethyl)amine, TBTA). Further,ascorbic acid reduces Cu (II) to Cu (I) and TBTA(tris(benzyltriazolylmethyl)amine) protects Cu(I) from oxidation anddisproportionation, while enhancing its catalytic activity.

A schematic of the CLICK reaction that conjugates a U1 Adaptor to cRGD₂is shown below. For simplicity, most hydrogens are not shown and the U1Adaptor is shown in a 3′ to 5′ orientation. The 3′ di-thio present onthe U1 Adaptor allows for either 1) attachment to other moieties or 2)reduction to leave a free —SH group, which has been shown to enhance invivo efficacy (see, e.g., Torres et al. (2012) Trends Biotech.,30:185-190).

The click coupling was performed in different amounts to demonstrate thescalability. In one reaction 20 μl 0.5 mM Hex-BCL2-A-SS U1 Adaptor, 10μl 2M triethyl amine acetate (TEAA) pH 7.0, 50 μl DMSO, 6 μl 5 mM(cRGD)₂-PEG₄-azide, 1 μl 50 mM ascorbic acid, and 8 μl water werecombined. The solution was degassed with argon and then 5 μl of 10 mMCuSO₄-TBTA (109 μl 0.1M TBTA, 441 μl DMSO, 500 μl 5 mg/ml CuSO₄) wasadded. The solution was again degassed with argon and then incubatedovernight at room temperature. 2 μl EDTA was then added. The solutionwas then desalted (spin column). 108 μg of the final conjugate wasobtained. In the second reaction 220 μl 0.5 mM Hex-BCL2-A-SS U1 Adaptor,110 μl 2M TEAA pH 7.0, 550 μl DMSO, 66 μl 5 mM (cRGD)₂-PEG₄-azide, 11 μl50 mM ascorbic acid, and 88 μl water were combined. The solution wasdegassed with argon and then 55 μl of 10 mM CuSO₄-TBTA (109 μl 0.1MTBTA, 441 μl DMSO, 500 μl 5 mg/ml CuSO₄) was added. The solution wasagain degassed with argon and then incubated overnight at roomtemperature. 22 μl EDTA was then added. The solution was then desalted(spin column). 1183 μg of the final conjugate was obtained. As statedabove, the conjugates were pure as determined by 8% PAGE.

The peptide-Adaptor conjugates were tested in a C8161 human melanomasubcutaneous xenograft mouse model. Specifically, nude mice implantedwith C8161 subcutaneous xenografts were treated twice per week for 3weeks by intravenous administration of either BCL2-A U1 Adaptor incomplex with a cRGD-dendrimer vehicle at 1.7 μg Adaptor/dose or BCL2-AU1 Adaptor-cRGD conjugate (no dendrimer) at 6 μg conjugate/dose. As seenin FIG. 57, the peptide-U1 Adaptor conjugate was as effective as, if notmore effective than, the dendrimer-based delivery vehicle.

An anti-KRAS U1 Adaptor-iRGD peptide conjugate was also determined toarrest pancreatic tumor growth in vivo. The iRGD peptide is adisulfide-based cyclic RGD peptide having the sequence CRGDKGPDC and adisulphide bridge from C1-C9 (Sugahara et al. (2009) Cancer Cell16:510-20). The human pancreatic cancer cell line MIA PaCa-2, which hasa KRAS G12C mutation, was implanted into the flanks of NCR nu/nu miceand tumors were allowed to become established for about 9 days. Miceunderwent treatments twice per week on Day 1 when tumors were about 10mm³ in size (n=6 to 7 per group). Specifically, mice were treatedintravenously 4.5 weeks (2×/week) with anti-KRAS-3 U1 Adaptor-peptideconjugated through its 5′ end to either cRGD or iRGD peptide at 0.8mg/kg with no nanocarrier. As seen in FIG. 58, treatment withcRGD-KRAS-3 or iRGD-KRAS-3 resulted in significant reduction in tumorgrowth, particularly with the iRGD-KRAS-3 U1 Adaptor.

While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope and spirit of thepresent invention, as set forth in the following claims.

Several publications and patent documents are cited in the foregoingspecification in order to more fully describe the state of the art towhich this invention pertains. The disclosure of each of these citationsis incorporated by reference herein.

What is claimed is:
 1. A compound for inhibiting the expression of agene of interest comprising: a) at least one nucleic acid moleculecomprising an annealing domain operably linked to at least one effectordomain, wherein said annealing domain hybridizes to the pre-mRNA of saidgene of interest, and wherein said effector domain hybridizes to the U1snRNA of U1 snRNP; and b) at least one targeting moiety and/or cellpenetrating moiety, wherein said targeting moiety and/or cellpenetrating moiety is operably linked to said nucleic acid molecule. 2.The compound of claim 1, wherein said annealing domain is about 10 toabout 30 nucleotides in length.
 3. The compound of claim 1, wherein saideffector domain is about 8 to about 20 nucleotides in length.
 4. Thecompound of claim 1, wherein said effector domain and annealing domainare linked by a bond.
 5. The compound of claim 1, wherein said effectordomain and annealing domain are linked by a linker domain of about 1 toabout 10 nucleotides.
 6. The compound of claim 1, wherein said effectordomain comprises the sequence 5′-CAGGUAAGUA-3′ (SEQ ID NO: 1).
 7. Thecompound of claim 1, wherein said effector domain comprises the sequence5′-CAGGUAAGUAU-3′ (SEQ ID NO: 32).
 8. The compound of claim 1, whereinsaid effector domain comprises the sequence 5′-GCCAGGUAAGUAU-3′ (SEQ IDNO: 33).
 9. The compound of claim 1, wherein said nucleic acid moleculecomprises at least one nucleotide analog.
 10. The compound of claim 9,wherein said nucleotide analog is selected from the group consisting oflocked nucleic acids and 2′-O-methylnucleotides.
 11. The compound ofclaim 9, wherein said nucleotide analog is a phosphorothioate.
 12. Thecompound of claim 1, wherein said annealing domain hybridizes with atarget sequence in the 3′ terminal exon of the gene of interest.
 13. Thecompound of claim 1, wherein the effector domain is operably linked tothe 3′ end of the annealing domain, the 5′ end of the annealing domain,or both the 5′ and 3′ end of the annealing domain.
 14. The compound ofclaim 1, wherein said annealing domain comprises a stretch of at leastseven deoxyribonucleotides.
 15. The compound of claim 1, wherein said U1snRNA is a U1 variant snRNA.
 16. The compound of claim 1, wherein saidnucleic acid molecule and said targeting moiety and/or cell penetratingmoiety are conjugated via a linker.
 17. The compound of claim 16,wherein said linker is cleavable.
 18. The compound of claim 1, whereinsaid targeting moiety and/or cell penetrating moiety is operably linkedto the 3′ end, the 5′ end, or both the 5′ and 3′ end of the nucleic acidmolecule.
 19. The compound of claim 18, wherein said targeting moietyand/or cell penetrating moiety is operably linked to the 5′ end of thenucleic acid molecule.
 20. The compound of claim 1, wherein said nucleicacid molecule is operably linked to a first targeting moiety at the 3′end and a second targeting moiety at the 5′end.
 21. The compound ofclaim 1, wherein said nucleic acid molecule is also operably linked to achemotherapeutic agent.
 22. The compound of claim 1, wherein saidnucleic acid molecule is operably linked to a targeting moiety and cellpenetrating moiety.
 23. The compound of claim 1 further comprising atleast one additional agent selected from the group consisting of adetectable agent, a therapeutic agent, a carrier protein, and agentswhich improve bioavailability, stability, and/or absorption.
 24. Thecompound of claim 1, wherein said targeting moiety is an antibody orfragment thereof.
 25. The compound of claim 1, wherein said targetingmoiety comprises an Arginine-Glycine-Aspartic Acid (RGD) peptide or ananalog thereof.
 26. The compound of claim 1, wherein said RGD analog iscyclic RGD (cRGD) or internalizing RGD (iRGD).
 27. The compound of claim1, wherein said targeting moiety is an oligonucleotide aptamer.
 28. Thecompound of claim 1, wherein said gene of interest is an oncogene. 29.The compound of claim 28, wherein said oncogene is a member of theB-cell lymphoma 2 (bcl-2) family or glutamate receptor 1 (grm1) or Kras.30. The compound of claim 29, wherein said member of the bcl-2 family isselected from the group consisting of bcl-2, bcl-XL, bcl-w, mcl-1,bfl1/A-1, and bcl-B.
 31. A composition comprising at least one compoundof claim 1 and at least one pharmaceutically acceptable carrier.
 32. Thecomposition of claim 31, wherein said composition further comprises atleast one siRNA or antisense oligonucleotide directed against said geneof interest.
 33. A method of inhibiting the expression of a gene ofinterest comprising delivering to a cell at least one compound ofclaim
 1. 34. The method of claim 33, wherein at least two of saidcompounds are delivered and wherein the annealing domains of saidnucleic acid molecules hybridize with different target sequences in saidgene of interest.
 35. A method of treating cancer in a subject, saidmethod comprising administering at least one compound of claim 1 to saidsubject.
 36. The method of claim 35, further comprising theadministration of at least one chemotherapeutic agent or radiationtherapy.
 37. The method of claim 35, comprising the administration ofmore than one compound, wherein a first compound comprises an annealingdomain which hybridizes to the pre-mRNA of a first gene and a secondcompound comprises an annealing domain which hybridizes to the pre-mRNAof a second gene.
 38. The method of claim 35, comprising theadministration of more than one compound, wherein a first and a secondcompound comprise annealing domains which hybridize to the pre-mRNA ofsaid gene.
 39. The method of claim 35, further comprising theadministration of at least one siRNA or antisense oligonucleotidedirected against said gene.
 40. The method of claim 35, furthercomprising the administration of at least one siRNA or antisenseoligonucleotide directed against a second gene.
 41. The compound ofclaim 1, wherein said cell penetrating moiety is an oligonucleotideaptamer.
 42. The compound of claim 41, wherein said oligonucleotideaptamer is C1.
 43. The compound of claim 1, wherein said cellpenetrating moiety is a nonpolar fluorescent group.
 44. The compound ofclaim 43, wherein said nonpolar fluorescent group is Cy3 or Cy5.